ROLE OF DECELLULARIZED LIVER MATRIX ON HUMAN EMBRYONIC STEM
CELL DIFFERENTIATION INTO FUNCTIONAL HEPATOCYTES
Nataly Carneiro Lessa
B.S., California State University, Sacramento, 2007
PROJECT
Submitted in partial satisfaction of
the requirements for the degree of
MASTER OF ARTS
in
BIOLOGICAL SCIENCES
(Stem Cell)
at
CALIFORNIA STATE UNIVERSITY, SACRAMENTO
SPRING
2011
ROLE OF DECELLULARIZED LIVER MATRIX ON HUMAN EMBRYONIC STEM
CELL DIFFERENTIATION INTO FUNCTIONAL HEPATOCYTES
A Project
by
Nataly Carneiro Lessa
Approved by:
__________________________________, Committee Chair
Thomas R. Peavy, Ph.D.
__________________________________, Committee Member
Jan A. Nolta, Ph.D.
__________________________________, Committee Member
Thomas E. Landerholm, Ph.D.
____________________________
Date
ii
Student: Nataly Carneiro Lessa
I certify that this student has met the requirements for format contained in the University format
manual, and that this project is suitable for shelving in the Library and credit is to be awarded for
the project.
__________________________, Graduate Coordinator
Susanne Lindgren, Ph.D.
Department of Biological Sciences
iii
___________________
Date
Abstract
of
ROLE OF DECELLULARIZED LIVER MATRIX ON HUMAN EMBRYONIC STEM
CELL DIFFERENTIATION INTO FUNCTIONAL HEPATOCYTES
by
Nataly Lessa
Liver disease is one of the leading causes of death worldwide, and over 25,000 people per
year die in the U.S. Often the only option available for liver disease patients is liver
transplantation, however, liver transplantation is severely limited due to the shortage of donors
and donor-recipient matches, especially for children. Hepatocyte transplantation is an alternative
for whole-organ transplants and it has been found to improve liver function. Unfortunately, there
is limited access to human hepatocytes and transplanted hepatocytes don't survive very long,
leading to problems with engraftment. Human embryonic stem cell (hESC) derived hepatocytes
have been sought as an alternative to primary hepatocyte transplantation. The goal of my project
was to test and improve hESC-derived hepatocyte differentiation protocols in order to obtain high
levels of mature hepatocytes in vitro. Since differentiation protocols vary in length, growth
factors used, and microenvironment for culturing, variables were tested to establish an improved
protocol for mature hepatocytes using a feeder-free system. Specifically, hESCs were induced to
form definitive endoderm (DE) before being matured into hepatocytes and whole organ
decellularized liver matrix (DLM) was examined as a carrier for hepatocyte transplantation.
The results showed that the differentiated hESC cells had the characteristic polygonal
hepatocyte morphology and were able to store glycogen. These differentiated cells secreted
albumin and synthesized urea by the end of the protocol (peaking at day 26) which is
characteristic of mature hepatocytes. The decellularization of the liver was improved by making it
iv
shorter than most decellularization processes and the residual DNA in the DLM was equivalent to
other protocols. The gross extracellular matrix shape and structure of the DLM was retained, as
shown by immunohistochemistry staining of key ECM proteins: laminin, fibronectin and collagen
IV. The effects of DLM on hESC derived hepatocyte survival were analyzed by infusing these
cells into the DLM after decellularization. Our data suggests that the DLM improves cell survival
in vitro. The DLM also supported cell survival in vivo for up to 7 days in an immunodeficient
mouse model. Furthermore, DLM seemed to be more effective in helping fetal hepatocyte cell
engraftment in vivo, as compared to splenic injections or Matrigel encapsulation, for up to 8
weeks, as evidenced by bioluminescence data. In addition, hESC derived hepatocytes were
examined to determine which integrins were expressed since this would inform us as to their
potential interactions with extracellular matrix proteins. It was found that the cells expressed
integrins 1, 3, 4, 5, V, 1, and 3. This suggests that these cells are capable of responding
to extracellular matrix proteins, including collagen, laminin and fibronectin. The identification of
these specific integrins provides a basis for understanding the mechanisms behind the effects of
liver ECM 3-D culture environment on hESC derived hepatocyte maturation.
In conclusion, this study resulted in an improved hESC-derived hepatocyte differentiation
protocol and established a short and efficient decellularization protocol for liver decellularization
in rodents. The DLM seems to help maintain hepatocyte differentiation in vitro, and it facilitated
cell engraftment and long-term survival in vivo when seeded with fetal hepatocytes. Overall, our
data suggests that DLM may be developed as an alternative carrier for hepatocyte transplantation.
__________________________________, Committee Chair
Thomas R. Peavy, Ph.D.
____________________________
Date
v
ACKNOWLEDGMENTS
I would like to thank all the amazing people who helped me put this project together. In
particular, I’d like to thank Dr. Ping Zhou and Dr. Thomas Peavy for being such a wonderful
mentors. Thanks to Dr. Jian Wu, for his mentorship, very detailed explanations and his love for
livers. Thank you Dr. Jan Nolta, for inspiring me to study stem cells with your talks and
excellence at work. I will be forever grateful to your mentorship in the field of regenerative
medicine. Thank you Dr. Nolta for the internship opportunity, for truly caring about every student
and every scientist at her lab, and for providing a state-of-the art lab facility, with many
opportunities for intellectual dialogue, which allowed me to grow as a scientist. A huge thanks to
all the members of the Nolta Lab, especially to Ella Severson for being my lab partner and
sticking with me through the long days under the hood. To Gaela Mitchell, Stefanos Kalomoiris
and Dr. Fernando Fierro for long weekends at the lab, which were only bearable due to your
smiles and laughter. A big thanks to my fellow graduate students at CSUS, in particular to
Heather Stewart, Michelle Ohlson and Ninnie Abrahamsson for listening to me whine, for
helping me at the lab and with our class projects. Thank you for your support and most
importantly for your friendship. Thank you to all the committee members: Dr. Nolta, Dr.
Landerholm, Dr. Lindgren and especially to Dr. Peavy for all his hard work and countless project
revisions that made this final document possible. “In essence, the bottom line is: ‘Measure twice,
cut once’ “. Thanks to all my friends who touched my life during the happy and difficult times. I
could not have completed the program without the love and support of my family: Mae, Pai,
Romulo and Cyro. Thank you for supporting me and encouraging me to follow my own path,
even when I decided to go so far away from home. Thanks to my fiancé Jakub, for all his support
and countless hours patiently waiting for me to be done with homework and lab work.
vi
TABLE OF CONTENTS
Page
Acknowledgments.....................................................................................................................vi
List of Figures ....................................................................................................................... .viii
List of Acronyms ......................................................................................................................ix
INTRODUCTION………………………….………………………………………….… ........ 1
MATERIALS AND METHODS .............................................................................................. 7
RESULTS ............................................................................................................................... 13
DISCUSSION ......................................................................................................................... 29
Literature Cited…………...……………………………….………………………………….36
vii
LIST OF FIGURES
Page
Figure 1. Characterization of DLM ……………………………………………….....….. 14
Figure 2. Characterization of hESC-derived hepatocytes (hEHs) ………….......…...…... 16
Figure 3. Functional Characterization of hESC-derived Hepatocytes (hEHs) …........….. 18
Figure 4. Gene expression levels of hESC-derived hepatocytes (hEHs)…….…….…….. 20
Figure 5. Functional Characterization of hEHs ….………………………….….……….. 22
Figure 6. DLM facilitates maintenance of hEHs in vivo………………….…….……….. 24
Figure 7. Bioluminescent imaging of Fetal Hepatocyte-infused DLM over time after
transplantation ….……………………………………………………….……... 26
Figure 8. Integrin expression in human embryonic-derived hepatocytes (hEHs)..…..…... 28
viii
LIST OF ACRONYMS
AAT – Alpha-1-Antitrypsin
AFP – Alpha-Fetoprotein
ALB – Albumin
ASGPG – Asialoglycoprotein Receptor
CYP – Cytochrome p450 proteins
D2 – Stage 2 of Differentiation
D3 – Stage 3 of Differentiation
DAPI – 40,6-diamidino-2-phenylindole
DE – Definitive Endoderm
DLM – Decellularized Liver Matrix
ECM – Extracellular Matrix
GAPDH – Gluceraldehyde-3-phosphate Dehydrogenase
GFP – Green Fluorescent Protein
H9 – Approved Human Embryonic Stem Cell Line
H&E – Hematoxylin and Eosin Stain
hEH – Human Embryonic Stem Cell Derived Hepatocytes
hESC – Human Embryonic Stem Cells
HGF – Hepatocyte Growth Factor
IL-2R-  -/- – Interleukin-2 Receptor Gamma
MEF – Mouse Embryonic Fibroblast Cells
mRNA – Messenger RNA
ix
MSC – Mesenchymal Stem Cells
NOD – non-obese diabetic
OSM – Oncostatin M
PBS – Phosphate Buffered Saline
RT-PCR – Reverse-transcriptase Polymerase Chain Reaction
SCID – Severe Combined Immunodeficiency
SDS – Sodium Dodecyl Sulfate
x
1
INTRODUCTION
Liver disease is one of the leading causes of death worldwide, and over 25,000 people per
year die in the U.S. (1). Often, the only option available for liver disease patients is liver
transplantation. However, liver transplantation is severely limited due to the shortage of donors
and donor-recipient matches, especially for children (2). Furthermore, transplanted patients have
to endure lifelong immunosuppression (3,4). Hepatocyte transplantation was sought as an
alternative for whole-organ transplants and found to improve liver function. Hepatocytes could be
used as a treatment option for liver disease patients. Still, limited access to human hepatocytes
and successful engraftment has been an issue (1). Therefore, a more abundant source of human
hepatocytes is desirable. In addition, hepatocytes could be used in basic and applied research as
well as in toxicity studies for drug development, since the liver is the main organ involved in the
metabolism of drug components (3, 5, 6, 7, 8).
Hepatocytes are parenchymal cells of the liver and account for 80% of the total cells in
the liver. The other 20% of the liver is composed of non-parenchymal cells such as sinusoidal
endothelial cells, stellate cells, Ito cells and Kupfer cells (9). Hepatocytes perform many functions
in the liver and are mainly associated with drug metabolism, synthesis of albumin, detoxifying
ammonia from the blood and production of bile salts (9). Due to the limited access to human
hepatocytes and problems with engraftment after transplantation, a renewable source of
functional hepatocytes is sought. As a result, many groups started using stem cell technologies in
order to circumvent this problem. Some groups suggest that there are liver specific stem cells but
these cells haven’t been isolated or characterized to date, and because there are other types of
cells in the liver, it would be difficult to define just one type of liver stem cell.
2
It is possible that stem cells from sources other than the liver could be used to produce
hepatocytes. Adult stem cells, such as Mesenchymal Stem Cells (MSCs) have been promising
candidates for regenerative medicine. MSCs are adult stem cells that can be easily isolated from
many tissues throughout the body including the bone marrow, adipose tissue, placenta, amniotic
fluid and umbilical cord blood. MSCs are easily cultured and have the potential to differentiate
into several cell types, including osteocytes, adipocytes, chondrocytes and myocytes. Some
groups reported that they can differentiate MSCs into hepatocytes when in the appropriate
microenvironment and stimulated with growth factors (9, 10, 11, 12). Therefore, MSCs were
considered good candidates for liver regeneration because their usage might overcome problems
with allograft rejection and immunosuppression since MSCs could be isolated from the patient
themselves (13, 14). However, our group and others have tried to differentiate MSCs into a
hepatocyte lineage with very limited success. Thus, the potential for MSC differentiation into
hepatocytes is still controversial.
Embryonic stem cells (ESCs) are still considered the best model for regenerative
medicine because ESCs are very primitive stem cells and have the potential to differentiate into
all cell lineages. However, human ESCs (hESCs) remain subject to ethical debate over their usage
and manipulation of human embryos. There are also safety concerns regarding potential germ cell
tumors (teratomas) being formed from poorly differentiated hESCs used in therapies.
Studies have shown that hESCs can differentiate into hepatocyte-like cells in culture but
with a success rate of only 10%. Furthermore, functional assays performed on these hepatocytelike cells demonstrated that these cells are not as mature as primary hepatocytes (5). Using
additional growth factors, seventy-percent of hESCs cultured in collagen type-I gels have been
shown to differentiate into hepatocyte-like cells (5, 6, 7). Gene expression assays suggest that
these cells not only express proteins similar to adult hepatocytes, but also fetal protein markers
3
such as α-fetal protein (AFP) which signifies that they are not fully matured, as evidenced by
functional assays (5, 6, 7).
Currently, there are many established protocols for hESC differentiation into hepatocytes.
hESC-to-hepatocyte differentiation protocols vary in length, growth factors used, and
microenvironment for culturing. Still, concerns over the functionality and maturity of these cells
drive the need to find better culturing conditions. Studies show that poorly differentiated hESCs
can form tumors in vivo in a mouse model (3). In an attempt to improve hESC-induced
hepatocyte differentiation, three groups used a similar protocol, in which hESCs were induced to
form definitive endoderm (DE) before becoming hepatocytes (5, 6, 7, 15). The formation of DE is
reminiscent of the liver embryogenesis process in vivo (5, 6). Priming hESCs to differentiate into
DE, before the addition of hepatocyte growth factors, showed an improvement in differentiation
yields (5, 6, 7). Furthermore, functional and gene expression analyses confirm that these hESCs
follow the in vivo sequential development of the hepatocyte cell lineage (5, 6, 7). Despite these
successes, studies have failed to quantitatively demonstrate that the expression of hepatocyte
markers by these hESC-differentiated hepatocyte (hEH) cells was comparable to physiological
expression levels in vivo (5, 6, 7).
Two of the challenges surrounding the use of adult hepatocytes are that, regardless of
their source, they are difficult to culture in vitro, and they tend to lose their function and dedifferentiate into more primitive cells (1, 8). In order to increase hepatocyte viability and
maintain functionality in vitro, several groups have used specialized cell culture substrates
containing collagen type-I, laminin and fibronectin. These protein substrates mimic the native
extracellular matrix (ECM) and provide support necessary for cells to adhere, spread, grow and
proliferate (1, 8). ECM-like substrates helped preserve the morphology and characteristics of
hepatocytes, but only for a few days (1, 8).
4
A sandwich configuration using these ECM-like substrates has been widely used for
hepatocyte culture. This constitutes a cell layer in between collagen type-I substrate or a
combination of collagen type-I with Matrigel®, made from ECM obtained from neoplastic cell
cultures (1, 16). The sandwich configuration improves hepatocyte morphology, polarity and
functionality over the single layer substrates, but it is far from ideal (1). Furthermore, solid nonbiodegradable and biodegradable substrates have been developed for hepatocyte culture, such as
polyurethane, polyglycolic acid and poly-L-lactic acid. Although the use of these solid substrates
was an improvement over single layer and sandwich model gels, there were significant
disadvantages with each of these substrates as they showed low cell viability and problems with
biocompatibility or biosafety, therefore making these cells unsuitable for clinical applications
(16). Due to the complexity of the three-dimensional structure of the liver, it is likely that more
specialized scaffolds would be needed to further improve cell maturation, support cell growth and
retention of cell functionality. The ideal scaffold would support cell attachment, migration,
differentiation and self-assembly into groups of cells (9).
Seeking an ideal substrate for hepatocyte growth, several groups started using tissueengineering methodologies. The concept of tissue bioengineering was developed as a treatment
option for patients with tissue loss. Tissues such as skin, cartilage and bone have been
successfully bioengineered and are now commercially available (8). Cells, scaffolds and bioactive
factors are typically used.
Recently, substrate gels made from decellularized, lyophilized rat and porcine liver tissue
proved to be successful in supporting the growth and functionality of hepatocytes (1, 16, 17, 18).
It is thought that native liver proteins and the 3-D interaction between cells and matrix allowed
for improved hepatocyte growth. Supporting the importance of the native ECM in cell growth
and functionality, hearts decellularized by coronary perfusion using SDS detergent showed that
5
this acellular matrix supported cardiac cell growth and functionality (4). Other groups have used a
similar technique and were successful at obtaining and re-seeding decellularized lungs, kidneys
and other organs with various types of native cells. These decellularized organ matrixes showed
great promise for such tissue engineering studies and could be applicable for liver bioengineering.
Therefore, we hypothesized that a decellularized liver matrix should provide an ideal
environment for the differentiation and retention of functionality of hESC-induced hepatocytes
(hEHs) as compared to hEH cells transplanted via splenic injection, the currently used method.
Despite all the advances in hESC differentiation into hepatocytes using different
protocols and microenvironments, studies have failed to quantitatively demonstrate that hESCderived-hepatocyte cells expressed hepatocyte markers that were comparable to physiological
expression levels in vivo (5, 6, 7). Therefore, DE-induced hESC differentiation, in combination
with cell culture in decellularized liver ECM using serum-free medium could improve hESC
differentiation into hepatocyte-like cells with enhanced functionality.
In this study we used decellularized mouse liver as a support matrix for hESC-derived
hepatocyte culture in serum free conditions as well as transplantation analyses in vivo. The goal
of my project was to test and improve several hESC-derived hepatocyte differentiation protocols
in order to obtain high levels of mature hepatocytes in vitro. In order to analyze these cells and to
determine different stages of differentiation for morphology and functionality, immunostaining of
hepatocyte markers, q-RT-PCR for hepatocyte markers, as well as functional analyses based on
albumin production, urea secretion and glycogen storage were performed. Premature hepatocytelike cells and late-stage hepatocyte differentiation cells were perfused into decellularized liver
matrix for in vitro studies as well as transplantation into the non-obese diabetic- severe combined
immune deficient, interleukin 2 gamma knockout (NOD/SCID/IL2r -/-) mouse model for in vivo
assessment. The cells were monitored for survival. In addition, the expression levels of AFP,
6
albumin (early fetal liver markers) and AAT (Alpha-1-anti-trypsin), cytochrome P450 enzymes
CYP1A1, CYP2C9, CYP7A1 and CYP3A4 (early and mature hepatocyte markers) were assessed
by quantitative RT-PCR.
7
MATERIALS AND METHODS
This research internship took place at Dr. Jan Nolta’s translational research laboratory under the
supervision of Dr. Ping Zhou at the University of California Davis, Institute for Regenerative
Cures in Sacramento, CA. This research project was conducted from June 2010 until December
2010. This project was funded by the California Institute for Regenerative Medicine (CIRM).
hESC Cell Culture
Human embryonic stem cells (H9) were purchased from WiCell Research Institute (Madison,
WI), cultured and propagated in gelatin-coated plates with mouse embryonic fibroblasts (MEFs)
as a feeder layer in standard hESC culturing media (Knockout DMEM/F-12 supplemented with
20% Knockout replacement serum, 2mM glutamax, 0.1M Non-essential amino acids, 0.1mM
beta-mercapto-ethanol, 1% penicillin/streptomycin and 8ng/mL bFGF). After reaching high
confluency, cells were transferred to Matrigel® (BD) coated plates with mouse embryonic
fibroblast conditioned medium (MEF-CM) in basic fibroblast growth factor in feeder-free, serum
free conditions as described (5).
hESC differentiation into Definitive Endoderm
hESCs were cultured in RPMI 1640 medium (Invitrogen) supplemented with 50-100 ng/mL
Activin A, 1mM sodium butyrate (NaB) and 1% L-glutamin for 1-7 days depending on the
protocol. Following, cells were cultured in the same medium, except for reduced concentration of
NaB to 0.5mM, and addition of 1xB27 (5).
8
Definitive Endoderm differentiation into Hepatocyte lineage
Cells were split with trypsin and re-seeded on collagen-I-coated plates. Cells were cultured in a
maturation medium (D2 = IMDM enhanced with 20% FBS, 2 mM L-glutamin, 0.3 mM 1thioglycerol, 1% antibiotic-antimycotic, 0.5% DMSO, 0.126 U/mL human insulin, 100nM
dexamethasone, 20ng/mL FGF-4, 20ng/mL HGF, 10ng/mL BMP-2 and 10ng/mL BMP-4 for 1014 days) unless otherwise stated. Following, cells were treated with (D3) Hepatocyte Culturing
Medium (HCM) supplemented with SingleQuots (Lonza, Walkersville, MD) and enhanced with
2-5% FBS, 20ng/mL FGF-4, 20ng/mL HGF, 50ng/mL Oncostatin M, 100nM dexamethasone and
0.5% DMSO for 8-12 days. Cell culturing medium was changed every 48 hours. Cells to be
implanted in the DLM were transduced with a lentiviral LUX-PGK-EGFP vector with firefly
luciferase and green fluorescent protein genes, at 20 multiplicity of infection (MOI) in the
presence of protamine sulfate (8ug/mL).
Whole liver perfusion decellularization
All animal experiments with performed in accordance with the NIH guidelines. The Institutional
Animal Care and Use Committee (IACUC) approved all animal protocols. Mouse livers were
decellularized following a previously described perfusion procedure with modifications (4, 7).
Briefly, mice were anaesthetized intraperitoneally with Pentobarbital (60mg/kg) or 100 mL/kg
ketamine with 10 mg/kg xylazine. Mice were fixed on an operation board and an u-shaped
transverse incision in the abdomen was made. The intestines were displaced to the left of the
abdominal cavity and a loose knot around the hepatic portal vein was made. The catheter was
inserted into the portal vein and perfusion tubes were connected. The cells were removed
chemically from matrix by perfusing with heparinized PBS (12.5 U heparin/mL) for 15 minutes,
0.1-1% SDS for 2-4 hours, followed by 30 minutes of 1% Triton-X100 in deionized water. The
9
decellularized liver matrix was perfused with PBS for 3 hours following the detergent treatment
to wash away residual SDS. The matrix was then perfused with the appropriate medium for an
additional 10 minutes prior to infusion of cells. Perfusion procedures were performed at 37ºC at
5mL/minute perfusion speed (4, 19).
Re-seeding decellularized mouse liver with cells
After decellularization procedure, the decellularized liver matrix was perfused with cell culturing
medium at 37ºC to establish the presence of nutrients and a physiological pH in the DLM. The
residual DNA content of the DLM and fresh mouse livers was then measured. Briefly, the DLM
and fresh liver were minced and DNA was extracted using a Qiagen Genomic DNA extraction kit
and quantitated using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE).
In order to visualize the vascular network, crystal violet dye dissolved in 1% low melting agarose
was injected via the portal vein. Micrograph images of the retained vasculature were taken (4,
19).. For cell seeding, 1-2 x 106 hESC-derived hepatocyte cells or human primary hepatocytes
(hPH) suspended in cell culturing medium were delivered via catheter with a 1mL syringe. The
liver was removed from the animal body, and lobes cut into small pieces. Seeded DLM pieces
were cultured in 24-well plate with HCM, frozen in OCT and/or used for transplantation.
Transplantation of DLM into mouse models
Non-obese, Severe combined immunodefficient, IL-2 gamma receptor negative
(NOD/SCID/IL2-/- or NOD/SCID/MPSVII mice) from Jackson Laboratories, Bar Harbor,
Maine, were bred at the UC Davis animal facility in compliance with all regulatory committees.
Mice were anaesthetized as previously described and fragments of the seeded DLM (approximate
size 0.5 x 0.5 x 0.1 cm) reconstituted with hESC-derived hepatocytes, fetal or primary
10
hepatocytes were implanted into the peritoneal cavity of the mice by suturing the DLM into the
omentum. The control mice were transplanted with 1 x 106 human primary hepatocytes or fetal
hepatocyte cells in 100uL of medium via splenic injections. The second control group received
transplantation of hepatocytes after Matrigel encapsulation (1 million cells in 100 uL of 25%
Matrigel in medium into the omentum).
Bioluminescent imaging of transplanted animals
Transplant recipient mice were injected with D-luciferin potassium salt intraperitoneally (150
mg/kg body weight in 100 uL PBS) and were imaged under isofluorane anesthesia with IVIS 100
Imaging System (Xenogen Corp) at the Center for Molecular and Genomic Imaging at UC Davis.
Recipient mice were imaged 24 hours following the procedure and once a week after that for up
to eight weeks. Mice were imaged for 5 minutes each time. Bioluminescent intensity was
quantified in units of maximum photons per second per centimeter squared per steradian
(p/s/cm2/sr) with Living Image software (20). After eight weeks following transplantation, mice
livers and the implants were harvested, cut into smaller pieces, snap frozen, and embedded in
OCT (optimal cutting temperature) compound. The frozen sections were stored in liquid nitrogen
until used. hEH cells harvested from the liver constructs in the animals were analyzed by
immunohistochemisty, and RNA was isolated for qRT-PCR as described later. The control
animals received hESC-derived hepatocytes (hEH) or fetal hepatocytes transplanted via splenic
injections only, hEH or fetal hepatocytes embedded in Matrigel or received no treatment.
Immunohistochemistry and Immunofluorescence
Cells and DLM tissues were fixed in 4% paraformaldehyde with PBS for 20 minutes, washed
with PBS, and permeabilized with 0.2% Triton-x-100 in PBS for 30 minutes. The sections were
11
blocked with 1% BSA for 1 hour. Cells were incubated with primary antibody at 4ºC overnight.
Cells were washed with PBS and incubated with a secondary antibody conjugated with Alexa 488
(Invitrogen) for 1 hour. After a PBS wash, cells and fragments were mounted with mounting
medium containing DAPI (Vector Laboratories). The DLM sections were also stained with
hematoxylin and eosin to examine cell components. Cells were stained for human albumin
(ALB), alpha-fetoprotein (AFP) and SOX-17 for endoderm. For in vivo studies, the same
procedures were used to analyze the extracted tissue (7).
Human Albumin secretion assay to evaluate liver function
Cell culture media was analyzed for human albumin content. Cell culture supernatant was
collected every 48 hours starting at the second stage of differentiation (D2). Quantitation of the
human albumin content was performed using a commercially available Human Albumin ELISA
Quantitation kit (Bethyl Laboratories Inc) and normalized to total cell number using a
hemacytometer (7).
Urea Synthesis Assay
Cell culture supernatant was collected every 48 hours starting at the second stage of
differentiation (16). Urea synthesis was measured using a commercially available kit from
Catachem (Urea diagnostic kit). Briefly, urea nitrogen reagent was added to the culture medium.
Urea nitrogen concentration in the medium was determined after a 60 and 120 seconds incubation
period. The urea nitrogen concentration was measured using a spectrophotometer. The urea
nitrogen concentration (mg/dL) in the samples was calculated as described in the manufacturer’s
protocol.
12
Periodic Acid-Schiff Assay for Glycogen
hEH cells were fixed with 4% parafolmaldehyde (PFA) and stained using a Periodic Acid- Schiff
(PAS) staining system from Sigma-Aldrich, according to the manufacturer’s instructions. Cells
were counterstained with hematoxylin.
Reverse-Transcription and quantitative Real-Time PCR
Total RNA was isolated from cells using RNeasy Mini Kits from Qiagen. Reverse-transcription
PCR was performed using One-step RT-PCR kit and random hexamers as primers from AB
Biosciences under default conditions (6). Quantitative RT-PCR was performed using ABI Prism
7300 Sequence Detection System using primers conjugated to FAM based probes for the
following genes: Albumin, AAT, CYP1A1, CYP7A1, CYP2C9, CYP3A4, and GAPDH. The
primer sequences are the same as listed in the following references (6, 7). The relative expression
of each gene was normalized to GAPDH, the reference gene.
Statistical Analysis
Bioluminescent intensity was expressed as mean +/- SEM. In vitro RT-PCR data were analyzed
by an unpaired student t test. In vivo RT-PCR data was expressed as mean value.
13
RESULTS
The experiments performed were designed to determine whether a decellularized liver matrix
(DLM) provides an ideal microenvironment to aid hESC-induced hepatocyte (hEH)
differentiation in vitro and whether the DLM can serve as a solid scaffold for hEH transplantation
into an animal model.
As a first step, the liver decellularization process was examined to optimize the potential
benefits of the DLM on hEH differentiation. To create a whole organ decellularized liver matrix,
we modified a previously established heart decellularization protocol (4). Mouse liver was
perfused in situ with the detergents SDS and Triton-X-100. SDS is an anionic detergent able to
penetrate through the cell membrane and solubilizes cell components (21). As the liver was
perfused with detergents through the hepatic portal vein, cellular components were removed from
the matrix. Upon treatment with SDS, the liver went from a red coloration to yellow within a few
minutes of treatment. As the process continued, the liver gradually started to lose color, until it
became translucent and gelatinous. The gross shape of the liver was retained however there was a
slight change in volume. Figure 1A shows the time sequence of the process of decellularization
using 1% SDS treatment (up to 120 minutes). Following SDS treatment, the liver was treated
with 1% Triton-X-100 and washed with PBS in order to remove excess SDS from the matrix.
After decellularization, livers were extracted from animals, cryopreserved, sectioned for
immunostaining, and then analyzed (Figure 1). Hematoxylin and eosin staining of decellularized
livers showed that there were no intact cellular nuclei or cellular components in the DLM (Figure
1C). The residual DNA obtained from the DLM was only 4% of the normal mouse liver DNA (73
+/- 39 ug/g DLM as compared to 1750 +/- 291 ug/g whole liver, respectively). The structure of
the liver was well preserved as shown by the extracellular matrix proteins such as collagen IV,
14
Figure 1. Characterization of DLM. (A) Mouse liver images during decellularization process over
time. (B) DLM harvested. (C) H&E staining of a DLM slice (x100). (D) Mouse liver and the
DLM cryosections were immuno-stained for the indicated extracellular matrix proteins (green)
and DAPI, blue (x200). (E) DLM was injected with crystal violet for visualization of the
vasculature (x20).
15
laminin and fibronectin, which stained positive in the DLM, an indication that the
decellularization process does not affect these important proteins (Figure 1D). DAPI staining of
the normal liver and DLM showed that there were no intact cell nuclei in the DLM. The liver
vascular system was also well preserved and could be seen without a microscope. To show that
the vasculature of the liver was intact, we visualized the vascular bed by injecting crystal violet
via the portal vein (Figure 1E). The dye flowed from larger vessels to smaller capillaries, as
expected. Thus, the liver was successfully decellularized without affecting the properties of the
matrix/structure of the liver itself, as indicated by immunostaining for key ECM proteins. This
protocol seemed to remove cellular components without affecting the quality of the matrix.
In order to differentiate hESCs into a hepatocyte lineage, we used an established protocol
developed by our collaborators, which was further optimized for H9 hESC differentiation into
hepatocytes (22). Slight modifications were made to the protocol, including starting
differentiation in feeder-free conditions using Matrigel. Matrigel was used as opposed to mouse
fibroblast feeders (MEFs) in order to develop a differentiation protocol more suitable for future
translational research. It was observed that Matrigel supported hESC-differentiation just as well
as mouse fibroblast feeders. hEH cells cultured on Matrigel had a more homogeneous population
of cells at the end of the differentiation protocol, as compared to cells on mouse feeder cells. In
addition, hEH cells cultured on Matrigel have similar gene expression patterns as hEHs cultured
on mouse feeders, as analyzed using qRT-PCR (data not shown).
A three-step differentiation protocol was utilized starting with hESC differentiation into
Definitive Endoderm (DE) during the first 5-7 days, followed by differentiation into the
hepatocyte lineage (8-12 days), and finally differentiation into mature hepatocytes during the last
5-10 days (Figure 2A). This protocol mimics the developmental process naturally occurring
during embryogenesis. This protocol attempts to recreate development by using known growth
16
Figure 2. Characterization of hESC-derived hepatocytes (hEHs). (A) Schematic representation of
the 3-step differentiation of hepatocytes from hESCs. (B-D) Immunofluorescent staining of hESC
derived Definitive Endoderm (DE) for DE marker Sox-17 (x100). (E-G) Staining of hEHs at Day
26 for hepatocyte markers ASGPR and ALB (x200).
17
factors to direct cells into the hepatocyte lineage pathway. At the beginning of the second step of
differentiation, a homogenous cell population was observed with uniform morphology, where
95% of the cells expressed DE specific protein marker Sox-17 (Figure 2B-D). Following
differentiation into DE, we converted these cells into hepatocyte progenitor cells that stained
positive for alpha-fetoprotein (AFP), a hepatocyte marker (AFP – data not shown). At day 26 of
the differentiation protocol (third stage), our cells expressed hepatocyte proteins such as
asialoglycoprotein receptor (ASGPR) and albumin (ALB), as shown by immunocytochemistry
staining (Figure 2E-G). Throughout the differentiation protocol, hESCs underwent a series of
morphological changes and the characteristic polygonal shape of hepatocytes was visible at the
second stage of the protocol. hEHs also showed a large cytoplasmic-to-nuclei ratio, and some
cells were found to have multiple nuclei and numerous vacuoles, normal to hepatocytes.
Hepatic function of the cells was assessed during the various stages of the differentiation
protocol. In order to test whether the hEH cells were secreting albumin, the culture media was
collected during the end of the second step and throughout the third step of differentiation. The
media was analyzed for human albumin content using a commercially available ELISA kit.
(Figure 3A-B). It was found that the hEH cells indeed secreted albumin into the cell culture
media and the levels peaked at day 26 of the differentiation protocol (50 ng/mL), and
subsequently declined. In addition, urea levels were measured from the medium since one of the
major liver functions is urea synthesis. Similar to albumin secretion, urea content peaked
between day 26 and day 28 (70ug/mL) of the differentiation protocol and then decreased by day
30. The amount of urea produced was comparable to human primary hepatocytes (Figure 3C).
Hepatocytes often store glycogen and liver glycogen is mobilized if the blood glucose levels fall
below normal. In addition, glycogen storage of hEH cells were examined by Periodic Acid Shiff
18
Figure 3. Functional Characterization of hESC-derived Hepatocytes (hEHs). (A-B) Albumin
secretion in media of hEH cultures at the indicated time points. (B) ALB secretion of hEHs and
human Primary Hepatocytes. (C) Urea production in hEH cultures at the indicated time points
and in hPH culture. (D) Glycogen storage in hEHs at Day 26 tested by PAS staining (x100).
19
staining (stains for carbohydrates). The cultured hEH cells were indeed capable of storing
glycogen, as seen by the purple-magenta coloration in Figure 3D.
To evaluate the hepatocyte specific gene expression profile of our hEH cells, we isolated
RNA from hEHs at the beginning of the differentiation protocol, at the end of the 2nd stage, and at
its completion. Quantitative RT-PCR was performed using GADPH to normalize the results and
hESCs were used as a negative control. Gene expression levels were compared to human primary
hepatocytes, hPH (Figure 4). Albumin expression was higher in hEH cells than in hPH (150%).
Albumin expression was not detectable in undifferentiated hESCs. Our hEH cells expressed some
hepatocyte genes at lower levels than in hPH, even at the end of the differentiation protocol;
alpha-anti-trypsin (AAT) was expressed at 40% of hPH, and undetectable on hESCs. The
cytochrome P450 proteins CYP3A4 and CYP2A1, which are involved in phase I of drug
metabolism in the liver, were expressed at 50% and 80% of hPH, respectively. CYP2C9, a
marker for mature hepatocytes had nearly negligible expression in hEH, comparable to hESC.
Also, a fetal hepatocyte marker, CYP7A1, was expressed much higher in hEH as compared to
hPH.
In order to further understand the effects of hepatocyte growth factor (HGF) and
Oncostatin M (OSM) on hEH maturation, culture conditions were altered at the 2nd and 3rd stages
of the protocol. We removed HGF and/or OSM from the culturing media. Albumin secretion and
urea synthesis levels of the hEH cells were analyzed and compared at various time points for the
different conditions, The currently established media protocol, including all growth factors, was
used as the control (Figure 5). The absence of HGF from culturing media has an impact in hEH
functionality, especially when HGH was removed at the 2nd stage of differentiation. In order to
test whether the hEH cells were secreting albumin and synthesizing urea, the culture media was
collected at day 23, day 25 and day 27 of differentiation, which under the established protocol,
20
Figure 4. Gene expression levels of hESC-derived hepatocytes (hEHs). Quantitative RT-PCR
analysis of indicated mRNA levels in H9s, hEHs at Day 20 and Day 30 in comparison to adult
Primary Hepatocytes (hPHs).
21
showed a peak in albumin secretion. The media was analyzed for human albumin content using
an ELISA kit.
Albumin secretion was significantly different in the absence of HGF, as compared to the
control conditions, especially when both HGF and OSM were absent from the culturing medium
at the 2nd and 3rd stage of differentiation, respectively (D2 and D3 stage). Albumin secretion was
five times higher in cultures in which HGF was not present at the 2nd stage, as compared to
control or cultures without OSM alone (Figure 5A). The effects of HGF were more pronounced
when HGF was absent at the 2nd stage rather than at the 3rd stage of differentiation, Albumin
secretion was eight times higher in cultures that did not have both HGF at the 2nd stage and OSM
at the 3rd stage. The absence of HGF or OSM alone at the 3rd stage of differentiation had little or
no effect on albumin secretion as compared to the control conditions.
Urea synthesis was also higher in the absence of HGF, as compared to the control, but not
as significantly as albumin secretion (Figure 5B). Urea synthesis was higher in culture conditions
omitting HGF at the 2nd stage than the control. In the absence of HGF or OSM at the 3rd stage of
differentiation (D3), urea synthesis was slightly higher than in the control conditions. The
absence of both HGF (at the 2nd stage) and OSM (at the 3rd stage) combined caused urea synthesis
to be slightly higher than in the control conditions, but not as high as in the complete absence of
HGF from the media (D2 and D3). There was no significant difference between the effects of
HGF on urea synthesis when it was omitted at the 2nd stage or completely omitted from the
culturing conditions.
Gene expression analysis was performed using the same culturing conditions as described
above but was found to be inconclusive (data not shown). Results showed no difference in
albumin or AAT expression between the different conditions, and CYP enzymes had negligible
expression in all culturing conditions, including the control. Although these results might suggest
22
Figure 5. Functional Characterization of hEHs. (A) Albumin secretion in media of hEH cultures
in the absence of HGF and/or OSM at different steps of the differentiation protocol and at the
indicated time points. (B) Urea production in hEH cultures in the absence of HGF and/or OSM at
different steps of the differentiation protocol and at the indicated time points. D2 – 2nd stage of
differentiation. D3 – 3rd stage of differentiation.
23
that lack of HGF has an impact in hEH maturation, more gene expression analysis experiments
need to be performed in order to obtain a more quantitative and reliable result.
After characterizing the hEH cells, they were tested to see whether they were capable of
surviving in a decellularized liver matrix (DLM). The hEH cells were transduced with a lentiviral
vector containing EGFP and firefly luciferase genes to facilitate imaging of these cells. Since the
DLM retained a vascular bed, hEH cells were infused through the hepatic portal vein and
monitored for hepatocyte engraftment as well as characterized for their functionality in vitro
before transplantation into an animal model. hEH cells were infused into the DLM in a single
infusion step and the recellularized DLM was removed from the animal, placed on a culture plate
with hepatocyte culturing media, and cultured for up to 5 days in the incubator under normal
oxygen conditions. The recellularized DLM showed even distribution of hEH cells after infusion
and after 5 days in culture, respectively (Figure 6A-B). After 5 days, the cells lost hepatocyte
morphology and began to die (data not shown). To evaluate whether the hEH cells in the DLM
constructs could survive in vivo, we implanted recellularized DLM constructs into the omentum
of immunodefficient mice (NOD/SCID/IL2r -/- strain). After 7 days following the
transplantation, bioluminescent imaging of these mice demonstrated that the hEH cells could be
detected and were still viable (Figure 6C). Control mice received transduced hEH cells via
splenic injections, the currently used method for delivery of cells. We were unable to detect hEH
cells in the control mice after 7 days post transplantation (data not shown).
To assess the metabolic activity of implanted hEH cells, human albumin secretion by
hEH cells was quantified. We collected mice serum and checked for human albumin content
using an ELISA kit. Human albumin was detected in the serum of mice transplanted with DLM
construct, although at low levels (data not shown). Human albumin was not detected in the serum
of mice that received hEH cells via splenic injections. hEHs embedded into the DLM maintained
24
Figure 6. DLM facilitates maintenance of hEHs in vivo. Human embryonic stem cell-derived
hepatocytes transduced with lentiviral LUX-PGK-EGFP vector were infused in DLM and
implanted into NOD/SCID/IL2rγ-/- mice or directly injected into the spleen of the mice (Data not
shown). (A, B) Fluorescent images taken at 40x after infusing hEH cells at day 1 (A) and day 5
(B). (C) Bioluminescent image of mouse implanted with DLM embedded hEHs expressing
luciferase at 1 week after transplantation shows that hEHs were viable. Human ALB secreted by
hEHs could be detected in the mouse serum (data not shown).
25
their hepatic function in vivo for at least 7 days. Gene expression analyses were not preformed on
the recellularized DLM construct hEH cells.
To evaluate whether the DLM supports the survival and function of fetal hepatocytes in
vivo, DLM was seeded with transduced human fetal hepatocytes and transplanted into the
omentum of immunodeficient mice (NOD/SCID/IL2r -/- strain). For comparison, primary
hepatocytes were injected into the spleen as a control. Splenic injections are the preferred method
for hepatocyte transplantation in mice (2, 3, 14). As an alternative control, a second group of
mice were injected with primary hepatocytes encapsulated into Matrigel and transplanted into the
omentum. This was necessary to eliminate the possibility that Matrigel matrix alone was
sufficient to maintain fetal hepatocyte viability in vivo. Bioluminescent techniques were used to
image the cells since it is a non-invasive approach to track the engraftment of cells to the matrix
and their proliferation. Animals were imaged 24 hours after transplantation and once a week
following the procedure, for up to 8 weeks. Figure 7 shows the bioluminescent imaging of three
representative mice with the different transplantation approaches at the specified time points:
decellularized liver matrix, splenic injections and Matrigel/omentum injections. The horizontal
line denotes the minimum signal intensity needed for imaging. The bioluminescent signal
decreased in the liver area of mice subjected to splenic injections and Matrigel injections much
faster than in mice transplanted with the DLM construct. Within 3 weeks of transplantation, there
was only a faint luciferase signal in the splenic injected mice. After 4 weeks, the bioluminescent
signal of mice submitted to splenic injections was only 0.39% of the initial level. The
bioluminescent signal in mice injected with Matrigel declined in a similar way to mice submitted
to splenic injection, although less rapidly. By week 5, the signal in the Matrigel injected mice had
decreased significantly, and it was only 0.92% of the initial intensity, while the mice transplanted
with the DLM construct had a strong signal by the end of the 8th week. At week 8, the
26
Figure 7. Bioluminescent imaging of Fetal Hepatocyte-infused DLM over time after
transplantation. After transduction with lentiviral vector containing LUX-PKG-EGFP, Fetal
Hepatocytes were either infused into DLM and then implanted into mice or transplanted via
splenic or omentum injection. (A) Representative bioluminescent images for the same mice over
time with three mode of transplantation. (B) Bioluminescent signal intensity for the mice with
splenic injections, omentum injection or DLM implantation at each time point. *P compares
DLM vs. Matrigel. P compares DLM and splenic injections. *P and P < 0.05, P < 0.01, ***
P < 0.005 and ****P < 0.001
27
bioluminescent signal in mice transplanted with DLM construct was 2.65% of the initial intensity.
The bioluminescent intensity of the DLM group at the time points was statistically different
(p<0.05-0.01) from the other two conditions. DLM enhanced the survival of fetal hepatocyte cells
in vivo.
To gain insight as to how hEH cells interact with the decellularized liver matrix, we
examined a panel of common integrins since cells interact primarily with the ECM through
integrins. Signals from the ECM are transmitted via protein-protein interactions and delivered to
the cells. These signals regulate many fundamental cell processes (23). We also analyzed the
expression of albumin in our hEH cells by immunocytochemistry to see if albumin positive cells
showed the expression of certain integrins. Integrins are heterodimeric cell surface receptors with
an alpha and a beta subunit. As shown in Figure 7, the hEH cells express integrins 1, 3, 4,
5, V, 1, 3. hEH cells also expressed 2, 6 and 2 integrins (data not shown). The
expression of the latter integrins was not specific to hEH cells expressing albumin. On the other
hand, the expression of integrins 1, 3 and 1, co-localized with albumin expression. Cells that
expressed integrins 4 and 5 also expressed high levels of albumin, however, not all cells
expressing albumin expressed the integrins. Cells that expressed V and 3 did not express high
levels of albumin. hEH cells are capable of responding to extracellular matrix proteins, including
collagen, via 11, laminin via 31 and fibronectin via 41 and 51.
28
Figure 8. Integrin expression in human embryonic-derived hepatocytes (hEHs). Immunostaining
of hEHs for the indicated integrins (green), Albumin (ALB, red) and DAPI nuclear stain (blue)
suggesting hEHs are capable of responding to various ECM proteins (x200).
29
DISCUSSION
The liver is the second largest organ in the body and it carries out a multitude of
functions, including drug metabolism, detoxification, albumin and urea synthesis. Although the
liver has great regenerative capacity, if extensive damage is caused, the liver loses the capacity to
regenerate itself. The existing treatment for liver failure is organ transplantation, and the shortage
of donors led scientists to consider the field of tissue engineering and whole organ constructs.
There have been significant advances in the field of tissue engineering, specifically the use of
decellularized extracellular matrices from organs to aid in cell transplantation, for example, liver,
lungs, bladder, intestines and hearts (4, 19, 21, 24, 25). A previous study reported using porcine
decellularized liver matrix (DLM) sheets for hepatocyte culture in vitro for up to 10 days (1).
This group showed that by culturing hepatocytes with DLM in a sandwich configuration, these
cells were able to retain function similar to hepatocytes cultured in a Matrigel sandwich.
Although this approach seems promising, by sectioning the DLM into sheets or freeze-drying the
organ, the three dimensional structure is lost and this may affect how the cells interact with the
extracellular matrix proteins. In addition, such approaches limit the use of the existing
vascularization of the liver to deliver oxygen and nutrients to the cells whereas the use of DLM
retains the 3D structure and vascularization making this approach more likely usable in the
clinical setting. Our approach in this project was to use a whole organ liver decellularization
technique, modified from a heart decellularization technique, as a cell carrier for transplantation
(4).
In this study we improved the decellularization process by making it shorter than most
decellularization processes: limiting it to 6 hours as compared to 72 hours (19, 24). The residual
DNA in the DLM was minimum and it was equivalent to other protocols (24, 25). We
30
demonstrated that the gross ECM shape and structure is retained, as shown by
immunohistochemistry staining of key ECM proteins: laminin, fibronectin and collagen IV. The
vascular bed of the DLM is also retained, an important factor for nutrient and oxygen delivery to
the cells. This protocol seemed to remove cellular components without affecting the quality of the
DLM.
However, further improvement of the decellularization process is needed to reduce the
residual DNA content and a need to establish a method for ex-situ decellularization is desirable in
contrast to the in situ method used in this study. We often had problems with contamination of the
DLM. The in situ decellularization could also decellularize parts of the stomach and intestines,
thus leading to a potential source of bacterial contamination. Some groups have used bioreactors
as a method for ex-situ perfusion decellularization of the organ (24, 25). The advantages of ex situ
decellularization using a bioreactor are the potential for process scale-up, increase in sterility, and
the ability to use larger animal livers to make DLM. The use of bioreactors would also lead to a
more controlled re-cellularization of the DLM.
In an attempt to improve current protocols for hESC-differentiation into hepatocyte-like cells
(hEH), differentiation was initiated using a feeder-free system. This was important since foreign
animal cells, such as mouse fibroblasts that are commonly used as feeders for hESC cell culture,
cannot be used for transplantations in the clinical setting. After cell culture in the presence of
growth factors, hEH cells were shown to have the characteristic polygonal hepatocyte
morphology and were able to store glycogen, one of the major functions of hepatocytes. hEH
cells secreted albumin and synthesized urea by the end of the differentiation protocol, peaking at
day 26 of the protocol but then decreased thereafter. This data indicated that after day 26 of the
protocol, the cells start to dedifferentiate. Therefore, an extension of the protocol may lead to loss
of hepatic function, rather than enhanced maturation.
31
By using the current protocol, hESCs were differentiated into a homogeneous population of
Definitive Endoderm cells and subsequently into functional hEHs. Still, gene expression analysis
of some mature markers such as CYP3A4 and CYP2C9 showed that these markers are not
expressed at the same levels as primary hepatocytes. In agreement with the hepatocyte-specific
functional data, our gene expression data suggests that hEHs obtained were not as mature as hPH
but much more mature than fetal hepatocytes, suggesting that these cells are still immature. Other
groups have reported similar immaturity of hEH cells (2, 3, 5-7, 10, 11, 26, 27, 28).
Thus, the differentiation protocol still needs further improvement, as there is still
variability in the cell population by the end of the third stage of differentiation. Different batches
of hEHs show different levels of homogeneity of the population in regards to morphology and
slightly different results in regards to functionality and gene expression patterns. Therefore, our
protocol needs to be improved to give a more reproducible homogeneous population of mature
hepatocyte cells.
In an attempt to understand how the various growth factors in hEH culture media affect hEH
maturation, the concentration of growth factors at various stages of differentiation was examined.
It was observed that the absence of HGF at a second stage of differentiation could improve
morphology of culture and metabolic functions of the hEH cells as evidenced by increased
albumin production similar to that of primary hepatocytes. The lack of HGF in the culturing
medium had a less pronounced effect on urea synthesis, although an increase of urea synthesis
was seen. Albumin secretion reached the highest levels when HGF and OSM were omitted from
the protocol at the second and third stage, respectively. Urea synthesis peaked in the absence of
HGF at the second stage of differentiation. The absence of HGF at the second step seemed to
have a great effect on hEH maturation, but not at the third stage, suggesting HGF may not be as
important for DE-directed hepatocyte differentiation as previously thought. However, gene
32
expression analyses of these cells were not conclusive and needs to be repeated. Although these
results might suggest that the lack of HGF has an impact in hEH maturation, more gene
expression analysis experiments need to be performed in order to obtain a more quantitative
result.
In order to understand the effects of DLM on hEH survival, hEH cells were infused
immediately into the DLM after decellularization and cultured under normal conditions. hEH
cells were cultured in the DLM for up to five days before they started to dedifferentiate and lost
hepatocyte morphology. This suggests that the DLM improves hEH cells survival in vitro. In a
separate experiment, the DLM was re-seeded with luciferase-transduced hEH cells and
transplanted into the omentum of the animal model within 24 hours. After 7 days post
transplantation, the bioluminescent signal was still detectable, suggesting that the hEH cells were
able to survive in the DLM in vivo for up to 7 days. Human albumin in the mice serum also
suggests that DLM was effective in helping cells survival. However, after 14 days post
transplantation, the bioluminescent signal had decreased significantly. More experiments need to
be repeated as the difference between hEH survival in the DLM and in Matrigel encapsulation
were not statistically significant (data not shown).
Since hEH cells may be too fragile to survive the stress of transplantation, we assessed
whether the DLM could aid fetal hepatocyte survival in vivo. The data shows that the DLM seems
more effective in helping cell engraftment in vivo than splenic injections or Matrigel
encapsulation, for up to 8 weeks, as evidenced by the bioluminescence data. Previous studies had
shown that DLM was able to aid hepatocyte survival for up to 24 hours, and this data shows that
these cells can survive much longer than previously reported (24).
Although fetal hepatocytes seem to survive well in DLM culture conditions, hEH cells do
not grow well in DLM in culture. This is possibly due to the toxic effects of SDS detergent
33
residues in the matrix. hEH cells are fragile when compared to fetal hepatocytes or other cells as
they are difficult to maintain in culture and die easily. To diminish the effects of residual DLM
SDS on the cells, a more thorough washing of the DLM prior to seeding with cells would be
desirable. In addition, seeding cells at their peak maturity (day 26) could also improve
engraftment. Furthermore, proper vascular system in the reconstituted DLM may be critical for
cell survival in the DLM. Currently, a small DLM construct transplanted into the omentum was
able to support cell engraftment and survival for up to 8 weeks. However, a scale-up procedure
using this technology to treat larger animals and humans would not be possible without an
established blood supply for the insert (21, 25, 29, 30). In addition, the seeding of several liver
cell types may help further mature the hEH cells. Previous studies have shown that pre-seeding
DLM with other endothelial cell types and cord-blood cells improves vascularization (29). The
choice of cells for co-culture, differentiation and maturation strategies for hEH cells and a proper
vascular system for delivery of nutrients and oxygen will need to be studied (21). Also, a
thorough investigation on the benefits of co-seeding different cell types with hEH should be
performed.
In an attempt to understand how hEH cells interact with ECM proteins, an investigation of
the pattern of integrins expression was conducted. Integrins may seem to play a role in the ECMcell interaction as suggested by a recent study (23). Previous studies have shown that the liver
undergoes matrix breakdown and remodeling following a partial hepatectomy and that integrins
serve as receptors and signal transducers between the cells and the ECM, signaling for cell
aggregation at sites of injury and tissue repair (23). It was found that hEH cells express integrins
1, 3, 4, 5, V, 1, 3, which co-localize with the expression of albumin. Albumin negative
hEH cells also expressed 2, 6 and 2 integrins. This suggests that hEH cells are capable of
34
responding to extracellular matrix proteins, including collagen, via 11, laminin via 31 and
fibronectin via 41 and 51. The identification of specific integrins responsible for matrix
protein-induced maturation of hEH cells would provide the basis for understanding the
mechanisms behind the effects of liver ECM 3-D culture environment on hEH maturation (30).
Also, upon binding to the ECM, integrins have been known to activate several cascade pathways,
including FAK, PLC-gamma, MAP kinase and PI3 kinase pathways (30). Thus, future studies
should focus on how these signaling pathways affect hepatocyte maturation.
The use of human embryonic stem cells in research is still very controversial. Many groups
have successfully reported the differentiation of induced pluripotent stem cells (iPSCs) generated
from fibroblasts, into hepatocyte-like cells (27, 31). Recently, a group reported using hepatocytes
to generate iPSCs and then differentiated these cells into hepatocyte-like cells (32, 33). By using
hepatocytes to make iPSCs, the epigenetics of the resulting iPSC cell might aid a more efficient
differentiation method into hepatocytes. In addition, the advantage of using iPSCs over hESCs is
the potential for creating patient specific cells for treatment of multiple diseases, including liver
disease. However, there are concerns to the use of iPSCs such as partial reprogramming of
somatic cells into iPSCs, the low efficiency of differentiation of these cells into hepatocyte-like
cells as compared to hESCs, and the expansion potential of these patient-specific cells in a Good
Manufacturing Facility (GMP). Human iPSC technology is still being developed and a number of
hurdles need to be overcome before its use in liver disease therapy becomes a reality (31). It is
likely that iPSCs will be the preferred cell source for hepatocyte differentiation in the future as
the iPSC technology field advances.
In conclusion, this study resulted in an improved hESC-derived hepatocyte differentiation
protocol and established a short and efficient decellularization protocol for liver decellularization
35
in rodents. The DLM seems to help maintain hepatocyte differentiation in vitro, and it facilitated
cell engraftment and long-term survival in vivo when seeded with fetal hepatocytes. Overall, our
data suggests that DLM may be developed as an alternative carrier for hepatocyte transplantation.
36
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