STUDY TO DIFFERENTIATE CLINICAL-GRADE HUMAN EMBRYONIC
STEM CELL LINES TOWARDS A HEPATOCYTIC LINEAGE
A Project
Presented to the faculty of the Department of Biological Sciences
California State University, Sacramento
Submitted in partial satisfaction of
the requirements for the degree of
MASTER OF ARTS
in
Biological Sciences
(Stem Cell)
by
Belinda Ann McCoy
SPRING
2012
STUDY TO DIFFERENTIATE CLINICAL-GRADE HUMAN EMBRYONIC
STEM CELL LINES TOWARDS A HEPATOCYTIC LINEAGE
A Project
by
Belinda Ann McCoy
Approved by:
__________________________________, Committee Chair
Thomas Landerholm, Ph.D.
__________________________________, Second Reader
Jan Nolta, Ph.D.
__________________________________, Third Reader
Christine Kirvan, Ph.D.
____________________________
Date
ii
Student: Belinda Ann McCoy
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
Ronald M. Coleman, Ph.D.
Department of Biological Sciences
iii
___________________
Date
Abstract
of
STUDY TO DIFFERENTIATE CLINICAL-GRADE HUMAN EMBRYONIC
STEM CELL LINES TOWARDS A HEPATOCYTIC LINEAGE
by
Belinda Ann McCoy
The liver is one of the most vital organs in the human body. Whole-organ
transplantation is the only established treatment for liver failure. Thousands die before
a suitable organ is found. Hepatocytes comprise over 70% of the liver’s mass and carry
out most of its specialized functions. The liver’s ability to regenerate itself from as
little as a third of its undamaged mass through proliferation of its remaining
hepatocytes has spurred interest in hepatocyte transplantation to failing livers, but
hepatocyte acquisition competes with whole-organ procurement. Mature hepatocytes
do not proliferate in vitro, and tend to lose their liver-specific characteristics. Human
embryonic stem cells (hESCs), capable of self-renewal and able to give rise to most
other cell types, could potentially provide an unlimited supply of hepatocytes. Through
careful monitoring and passaging, a number of hESC lines have been generated. Early
hESC differentiation methods led to the formation of embryoid bodies (EB), containing
a low percentage of cells expressing hepatic proteins. Differentiating hESCs first to
definitive endoderm before progressing them towards hepatocytes has resulted in the
iv
generation of hepatocyte-like populations with over 90% of the cells expressing mature
hepatocyte properties. Cultured cells are often visually evaluated for morphology, or
by immunohistochemistry (IHC) for stage- or tissue-specific markers, such as Oct4 in
hESCs or albumin for hepatocytes. But the ability to function equivocally to in vivo
counterparts or functionality of vital cellular structures like mitochondria, which
produce ATP, may also be measured. hESCs for use in human cell therapy, must be of
high quality to ensure genetic stability, and derived under xeno-free culture conditions
to eliminate risk of transmission of animal pathogens or immune reactions to animal
proteins in human patients. Commonly, hESCs are maintained in culture on a layer of
mouse embryonic fibroblasts (MEFs). Alternatives to MEFs may still contain animal
products or are more expensive. Six clinical-grade hESC lines have recently been
developed and are available to researchers. In this study, lines ESI-017 and EST-035
were found to proliferate well on MEFs and Matrigel at early passage, similar to H9,
but acquired genetic mutation beyond passage 60 and demonstrated decreased ability to
differentiate towards hepatocytes with increasing passage number and do not appear to
be able to match the hepatic functionality of H9-derived-hepatocytes nor primary
hepatocytes.
_______________________, Committee Chair
Thomas Landerholm, Ph.D.
_______________________
Date
v
ACKNOWLEDGEMENTS
I would like to give much thanks to by project advisors Dr. Hao Nguyen, Dr. Thomas
Landerholm, and Dr. Thomas R. Peavy, my mentor Dr. Mark A. Zern for accepting me
into his lab, and Jan Nolta, and Gerhard Bauer for making available their labs to our
program. Also a special thanks to Ben Tschudy-Seney for his thoughtfulness and
support, Dr. Xiaocui Ma for her gentle guidance, and Charles X. Wang for pushing me
to apply my knowledge, not simply giving me the answers. This project was made
possible through the support of the California Institute for Regenerative Medicine
(CIRM), the CSUS Biological Sciences Department, and the U.C. Davis Institute for
Regenerative Cures. I would also like to give my most grateful thanks to my dearest
friends Terry Robbins and Theresa Robbins for their patience and sacrifice in
supporting my efforts, my father Dwight McCoy for being there to give me support at
my darkest moments, and my sister Serena Framkena for all her encouragement and
belief in my abilities even when I doubted myself. Lastly, I would like to thank the
other members of the second-year cohort of CSUS stem cell graduate students who
supported me and selflessly gave of themselves to make this project possible.
vi
TABLE OF CONTENTS
Page
Acknowledgements ………………………………………………………….……….. vi
List of Figures ………………………………………………………………..…..…. viii
Chapter
INTRODUCTION ……….…………………………………………………………..... 1
METHODS…………………………………………………………………………… 17
Cell culture, expansion, and maintenance of cell lines ...…………………..... 17
Induction of hESC and iPSC lines to definitive endoderm ………………...... 18
Differentiation of hESC and iPSC-derived DE to hepatocytes………………. 18
Detection of stage-specific markers through immunohistochemistry
fluorescence microscopy …………………………………………………...... 19
Karyotyping of clinical-grade hESCs for genetic abnormalities ………….…. 20
ELISA quantification of albumin secretion by derived hepatocytes…………. 20
Induction of P450 cytochrome activity within derived hepatocytes ……….... 21
Quantification of ATP produced by derived hepatocytes and primary
hepatocytes…………………………………………………………………… 22
Evaluation of mitochondrial mass in derived hepatocytes by MitoTracker
Green FM staining……………………………………………………………. 23
Evaluation of mitochondrial mass of hESCs through derived hepatocytes by
immunohistochemistry fluorescence microscopy…..………….…………….. 24
RESULTS………………………………………………………………………..…… 25
DISCUSSION………………………………………………………………………… 50
Literature Cited……………………………………………………………………….. 59
vii
LIST OF FIGURES
Figure
Page
1.
Differentiation of iPSCs to derived hepatocytes ….…………………………. 26
2.
Morphological comparison of iPSC-derived and primary hepatocytes …..…. 28
3.
IHC staining for hepatic markers of iPSC-derived and primary hepatocytes.. 29
4.
ESI-035 hESCs on MEFS …………………….……………………………... 31
5.
Induction of ESI-035 to definitive endoderm ….……………………………. 32
6.
IHC staining of ESI-035 derived hepatocytes for hepatic markers …….……. 33
7.
DE induction and hepatocyte differentiation of ESI-035 hESCs on Matrigel,
passage 41+ ………………………………………………………………….. 36
8.
Karyogram of ESI-017 hESCs at passage “43” ……………………………... 38
9.
Induction to DE and hepatocyte differentiation of ESI-017…………………. 40
10.
Amount of albumin by ESI-017-derived hepatocytes ……………………….. 42
11.
CYP2C9 rifampicin induction assay of iPSC- and ESI-017-derived
hepaocytes……………………………………………………………………. 44
12.
Levels of ATP present in cell extract from iPSC and ESI-017- derived
hepatocytes …………………………………………………………………... 46
viii
1
INTRODUCTION
The liver is one of the most metabolically active organs in the human body.
Many of its functions are vital for life. The liver is responsible for drug detoxification,
albumin and bile secretion, glycogen and vitamin storage, and carbohydrate, urea, and
lipid metabolism (Kulkarni et al., 2006). The importance of proper liver function is
such that its loss can result in systemic effects as severe as sepsis and multiple organ
failure (Soltys et al., 2010). For patients with life threatening liver disease and liver
failure, as well as the majority of liver-based metabolic disorders, even if the disease is
the result of a single enzyme deficiency and the liver otherwise functions normally,
whole-organ liver transplantation is currently the only established treatment option
(Sellaro et al., 2010). However, the availability of donor livers is in short supply,
treatment is expensive, and whole-organ surgery carries great risk. The number of
people waiting for a donor liver far exceeds the number of livers available. Thousands
die before a suitable donor organ is found; many others are ineligible for
transplantation; and tens of thousands are never added to the wait list (Everhart et al.,
2009; Zern, 2010).
The liver is comprised of several cell types, but by in large, hepatocytes are the
primary functional cells of the liver and carry out most of the organ’s specialized
functions (Bhatia et al., 1999; Kulkarni et al., 2006). Hepatocytes comprise about 70%
of the liver’s mass. They are large cells, often characterized by the presence of many
lipid and glycogen containing vesicles, and double nuclei. The liver possesses the
unique ability to regulate gain and loss of its cells and carefully adjusts its liver-to-
2
body mass ratio to meet the functional demands of the body; a property exploited in
partial hepatectomy in the treatment of disease or reduction of cells in transplantation
of a large liver into a small recipient (Fausto, 2000). Remarkably, the liver can
regenerate itself from as little as a third of its undamaged mass. Regeneration of liver
hepatocyte mass usually occurs through proliferation of the remaining hepatocytes, not
tissue-specific stem cells, as in replacement of lost cells in most other tissues (Fausto,
2000; Oertel and Shafritz, 2008). This is particularly significant because hepatocytes
are differentiated cells that rarely divide and are usually in a quiescent state; yet appear
to retain a “stem-ness” quality (Oertel and Shafritz, 2008). Successive transplantation
and regeneration experiments have shown hepatocytes capable of at least 69 rounds of
replication, a number seemingly beyond the normal cell-division limit (Fausto, 2000).
How hepatocytes are able to avoid the limitations of telomere shortening and extended
replication has not yet been fully elucidated.
The regenerative ability of hepatocytes has lead to much interest and
investigation into transplantation of hepatocytes to a diseased or failing liver.
Hepatocyte transplantation offers a promising treatment alternative to whole organ
transplantation that could bridge the gap for many patients with liver disease (Sellaro et
al., 2010). Hepatocyte transplantation is far less invasive than whole organ transplant,
typically involving just injection of isolated hepatocytes into the liver via the hepatic
portal or the spleen (Soltys et al., 2010). In addition, transplantation can be performed
on severely ill patients, and is repeatable. Hepatocyte transplantation could be a
particularly attractive option for patients with liver-based metabolic disorders, where
3
only specific liver functions needs to be restored, or for patients ineligible for wholeorgan transplantation.
Proof-of-principle has been demonstrated effective in humans receiving donor
hepatocyte infusions (Fisher and Strom, 2006; Nussler et al., 2006). Many studies with
animal and human subjects have demonstrated hepatocyte transplantation can mediate
metabolic deficiencies and even reverse liver failure (Fisher and Strom, 2006; Nussler
et al., 2006; Soltys et al., 2010). But viable human hepatocyte acquisition competes
with whole-organ procurement, and thus they are difficult to obtain (Thomas et al.,
2010). Currently hepatocytes used for cell-based therapy primarily come from livers
rejected for whole-organ transplantation, or unused segments of donor livers, as in the
case of pediatric recipients who need only a small liver. Lack of an abundant source of
hepatocytes remains a major hurdle to the clinical application of hepatocyte
transplantation.
Culturing cells in vitro, outside the body, has allowed for the expansion of
many mammal cell types to usable numbers. Most are used for research purposes but
other cultured cells are already in clinical use, such as cultured skin for grafting
treatment of severely burned patients. The most common method of culturing cells
recovered from an organ or tissue is by separating the cells from the source, usually
through enzymatic or chemical digestion of the non-cellular extracellular matrix
(ECM). Once isolated, the cells are distribute over the surface of a culture dish coated
with an adhesive material, such as gelatin or collagen, and supplying them with culture
4
medium that best represents the aqueous environment of the tissue from which the cells
originated (Freshney. 2010).
Some cell types grow well under in vitro conditions, others do not. At best,
culturing conditions are an approximation of the in vivo environment. In vivo, cell-cell
and cell-extracellular matrix interactions are extremely important. Cells interact closely
with each other and the extracellular components of collagens, laminins, fibronectins,
growth factors, enzymes and signaling molecules; an environment that is neither static
in composition nor concentration. In order to grow and proliferate, cells need to attach
to a surface and spread out (Freshney, 2010). The coating of the culture plate provides
the cells a supportive surface conducive to their attachment. ECM components native
to the cell type are often used for the coating material, but it is often impossible or too
expensive to mimic the complexity of the in vivo environment in the culture medium,
which for many cell types is still poorly characterized (Freshney, 2010). Cells that rely
heavily on close association with other cells to maintain their identity and functionality,
generally do not fare well once removed from their tissue (Freshney, 2010; Li, 2007).
Mature hepatocytes are one such cell type: they are difficult to maintain in
culture, and because they generally do not proliferate in vitro, the cultures are shortlived. Hepatocytes have a lifespan of hours in suspension and 3-4 days in plated culture
(Li, 2007). Collagen I, III, and IV and fibronectin are the major constituents of the liver
ECM, but the ECM of the liver provides more than a simple scaffold to hold the cells
of the liver together. The ECM forms compartments and barriers within the liver and
between cell types, it regulates growth factors and its proteins act as signaling
5
molecules themselves. It sequesters nutrients and growth factors, releasing them when
needed and in the appropriate concentration (Rodés et al., 2007). The close association
between hepatocyte and ECM and nonparenchyme cells is vital. In vitro, isolated
hepatocytes tend to dedifferentiate. Although they adhere to the culture plate and
spread out, they soon lose many of their liver-specific characteristics and their
metabolic function declines (Bhatia et al., 1999; Thomas et al., 2010). Eventually the
cell nuclei undergo karyolysis (chromosomal dissolution) and the cells die.
Cryopreservation has allowed for prolonged storage of isolated hepatocytes, and has
found use in research purposes, but cryopreserved hepatocytes do not engraft and
function well after thawing (Li, 2007; Soltys et al., 2010). In order to further advance
the development of hepatocyte cell-based therapies, an alternate source of readily
available functional hepatocytes is needed.
Human embryonic stem cells (hESCs) could theoretically provide an unlimited
supply of viable hepatocytes for use in the treatment of patients with liver disease
(Thomas et al., 2010). hESCs are cells derived from the inner cell mass of the
blastocyst stage of embryonic development.
They are capable of continual self-
renewal and are pluripotent, and can give to all three embryonic germ layers:
endoderm, ectoderm, and mesoderm, from which tissue-specific stem cells arise, which
in turn give rise to progenitor cells that then differentiate into all the other somatic cell
types of the body (Oertel and Shafritz, 2008).
hESCs are fragile and finicky, difficult to maintain in culture because the
slightest imbalance in growth factors or stress will cause them to differentiate. High
6
confluency on the culture plate and colonies growing into each other will trigger
uncontrolled differentiation, and hESCs passaged into cell aggregates of too few cells
often will not adhere and proliferate. While it has been found that mouse embryonic
stem cells can produce colonies reliably from cell clumps as few as two or three cells,
and have been found to even be able to produce a colony from a single cell, human
stem cells more reliably produce colonies when passaged in clumps of around eight
cells (Ginis et al., 2004). hESCs are extremely sensitive to temperature, toxins, and
bacterial and fungal contaminants. Antibiotics/antimycotics added to the media has
helped rid cell cultures of infectious agents, although care in their use must be taken as
antibiotics have been noted to affect growth and differentiation of stem cells (Cohen et
al., 2006). hESCs must be housed in temperature, humity, and CO2 controlled
incubators, and the media must be refreshed daily to assure a constant supply of
nutrients and reduce the potential for differentiation induced by a toxic environment
(Freshney, 2010; Oh et al., 2005). Through careful monitoring and repeatedly
transferring the cell colonies to new culture plates to ensure that cell populations
remain small, a number of hESC lines have been developed and in culture in an
undifferentiated state (Mannello and Tonti, 2007; Mitalipova et al., 2003). Due to its
stability and intense characterization, the H9 hESC cell line developed by researchers
at the University of Wisconsin in the mid 1990’s, has become the workhorse of many
laboratories investigating the use of hESCs for potential therapeutic use (Thomson et
al, 1998).
7
Because hESCs have the potential to give rise to any cell type of the body, they
cannot be used directly for in vivo cell therapy, as in vivo implantation of hESCs have
the ability to—and it is considered to be a hallmark characteristic of stem cells—form
teratomas, tumors containing cells from all three germ layers (Agarwal et al., 2008;,
Dalgetty et al., 2009). Once a stable population of hESCs has been established, they are
then differentiated towards specific cell lineages through carefully timed exposure to
selected growth factors, cytokines and signaling molecules, mimicking the interaction
between the cells themselves and their neighboring cells and tissues during embryonic
development in which a cell’s fate (endoderm, ectoderm or mesoderm) depends on the
concentration, presence, absence and length of exposure to particular molecular cues
(Agarwal et al., 2008; Dalgetty et al., 2009).
Cell morphology is often the first line of evaluation in cell culture. Many cell
types have distinctive shape and size, nuclear-to-cytoplasm ratio, presence of distinct
nucleioli, and organelle number and distribution. hESCs are characterized as being
small, round, with high-nuclear-to-cytoplasm ratio and prominent nucleioi. The purity
of a cell culture, particularly hESCs, and the success of differentiation towards a
specific cell lineage is commonly measured through detection of stage-specific or
tissue-specific markers—proteins of structures produced by or expressed on or
structures within the cell—preferably found only on or within the specific cell type.
The quickest method of marker protein evaluation is through immunohistochemistry
(IHC) staining using fluorescent antibodies. The transcription factors Sox2, Oct4, and
Nanog—whose expression are influenced by LIF—as well as the membrane receptor
8
proteins SSEA3 and SSEA4, are commonly used to evaluate the pluripotency of hESCs
(Boyer et al., 2005; Thomson et al, 1998).
Early studies attempting to guide hESCs toward a hepatocyte lineage met with
limited success (Agarwal et al., 2008). Early culture methods of ESC differentiation
involved suspension in culture medium containing relevant growth factors and
cytokines resulting in the formation of cell aggregates called embryoid bodies (EB).
EBs exhibit regional differentiation as in the early embryo, containing cell types of all
three germ layers (Agarwal et al., 2008; Dalgetty et al., 2009). While EBs can be plated
onto matrix substrates and create the microenvironment required for hepatocyte
differentiation, the process results in the spontaneous formation of a heterogeneous
population of cell types, from all developmental stages, including undifferentiated
cells, with low percentage of the cells expressing proteins similar to mature hepatocyte.
Further, poorly differentiated cells too tend to form teratomas in vivo (Agarwal et al.,
2008; Dalgetty et al., 2009). To be of therapeutic value, human embryonic derived
hepatocyte (hEDH) populations must be homogenous. Later groups were able to
improve the numbers and homogeneity of their hepatocyte-like cell populations
through the use of cell sorting techniques to purify away cells displaying early hepatic
lineage markers from the EBs (Dalgetty et al., 2009).
In vivo, definitive endoderm (DE) gives rise to many of the major organs,
including the pancreases, thyroid, lungs and liver. Nodal, a member of the TGFβ
family of molecules, plays a pivotal role in the in vivo development of DE (Lee et al.,
2005). It has been found that treatment of cultured stem cells with media containing
9
Activin A, a molecule that mimics Nodal but is much easier to acquire, can lead to the
differentiation of stem cells to definitive endoderm, through the Nodal pathway and the
expression of transcription factors such as Sox17 and FoxA2, which turns on
differentiation genes (Boyer et al., 2005; Lee et al., 2005). FoxA2 itself is a
transcriptional activator for liver-specific genes, such as albumin (Boyer et al., 2005;
Lee et al., 2005). Without Nodal, cells are destined for a mesodermal fate (D’Amour et
al., 2005; Zaret, 2001).
Thus, several research groups found that by first differentiating hESCs to
definitive endoderm, mimicking embryonic developmental in vivo before they
progressed the cells towards a hepatocyte lineage resulted in a hepatocyte-like
population with over 80% of the cells expressing mature hepatocyte proteins and
metabolic capabilities (Agarwal et al., 2008; Cai et al., 2007; Dalgetty et al., 2009).
Only after the hESCs have been pushed towards DE are they then treated with
additional growth factors, including FGF, BMPs, EGF, HGF, TGFβ, TNFα and IL-6 to
push the DE down the hepatic lineage, and finally HGF in conjunction with oncostatin
M, to further promote hepatocytic differentiation towards mature hepatocytes.
Successful differentiation from DE to mature hepatocyte is marked by a notably rise
than decrease in the production of alpha-fetoprotein, the fetal form of albumin to true
albumin, measured through IHC. By evaluating various culture media and refining the
concentrations of growth factors, Duan and his colleagues were able to develop
optimized culturing conditions that further improved the progress of directing the
10
differentiation of hESCs to derived hepatocyte-like cells to over 90% (Duan, et al.,
2010).
Once a seemingly homogenous derived-cell population has been produced, it
must be further evaluated before use for research, and especially, clinical therapeutic
purpose. The derived cells need to demonstrate the ability to not only display markers
of cell-specificity, they must also be able to function equivocally to the in vivo cells
they are supposed to be differentiating into. For derived hepatocytes functional
evaluation invariably includes the ability to secrete albumin into the media, as albumin
is produced primarily by the liver (Duan et al., 2010). But demonstration of the ability
to metabolize various drugs is also a crucial liver function, particularly the potential of
p450 cytochrome enzymes involved in phase I and II drug metabolizing activity which
occur primarily to the liver (Chen et al., 2004; Duan et al., 2010; Runge et al., 2000).
Those of the phase I class, whose activity results in oxidation products are easy to
assess by ELISA, while the more complex phase II isoforms often require more
sensitive detection by reverse-phase liquid chromatography (Duan et al., 2010; Runge
et al., 2000).
It should be noted, that, in the Duan study, although their H9-derived
hepatocytes displayed high expression of the mature hepatocyte marker human alpha 1antitrypsin, a protein secreted by hepatocytes into the blood stream that functions as an
elastase inhibitor in the lungs, and IHC staining detected significant amounts of
albumin present in the cytocol of the cells, albumin secretion and metabolic enzyme
activity of said cells was significantly lower than that found in primary hepatocytes
11
(Duan et al., 2010). Thus, despite outward appearance, the deprived hepatocytes are
still not equivalent and further investigation is needed. One possible hypothesis as to
why derived hepatocytes do not perform at the level of primary hepatocytes is that they
do not produce ATP at the same rate. Being one of the most metabolically active
organs in the body, the liver has a high energy demand, in the form of ATP, to carry
out its functions (Karp, 2008; Mann et al., 2001). In most mature cells, the majority of
ATP is produced in mitochondria via oxidative phosphorylation (Karp, 2008). While
mitochondria expansion has been evaluated in several derived cell types, the functional
capacity of mitochondria in derived hepatocytes has not yet been evaluated (Chen et
al., 2008; Lonergan et al., 2007).
Mitochondria are double-membrane organelles that often depicted as isolated
crescent-shaped beans, which actually form a complex network between themselves
and other organelles, particularly the nucleus and endoplasmic reticulum. Each
hepatocyte contains around 2000 mitochondria (Karp, 2008). Mitochondria are best
known as the powerhouses of the cell, as they are the organelles responsible for
production of over 90% of all cellular ATP. But they also are involved in many other
cellular functions, including fatty acid, amino acid metabolism, and heme synthesis,
and activities specific to the liver. Mitochondria of hepatocytes are essential for the
urea cycle and bile synthesis, and the heme produced is an essential component of the
many cytochrome enzymes necessary for the liver’s drug metabolizing activities, and
increased ATP production is required for liver regeneration via hepatocyte proliferation
(Mann et al., 2001; Yamashina et al., 2010). A number of liver diseases are known or
12
suspected to be due in part to mitochondrial dysfunction, including mtDNA depletion
syndrome, a respiratory chain disorder and a known cause of liver failure in children;
NAFLD, a disease in which excess fat builds up in the liver; and hepatocellular
carcinoma, where there is a down regulation of oxidative phosphorylation and
upreguation of gylcolysis (Yamashina et al., 2010).
In in vivo development, the blastocyte, from which stem cells are derived, exists
in an oxygen-poor hypoxic environment, which is not conducive to ATP generation via
oxidative phosphorylation (Lonergan et al., 2007; Nesti et al., 2007).
Energy
production is achieved primarily through glycolysis, an anaerobic process that does not
require oxygen, thus, stem cell mitochondria are metabolically quiescent, producing
only low levels of ATP. They tend to be small and spherical, with underdeveloped
cristae, few in number and localized in a perinuclear arrangement (Lonergan et al.,
2007; Nesti et al., 2007). Upon differentiation of stem cells towards mature cell
lineages, several groups have found that changes in mitochondrial morphology and
activity mirrors that of in vivo development seen following implantation. Mitochondria
number and size increases, they assume an elongated form with numerous, distinct
cristae, and they form an extensive network throughout the cell cytoplasm (Chen et al.,
2008; Lonergan et al., 2006). Morphologic change is accompanied by a several-fold
increase in ATP production to meet the cellular needs of mature cells (Lonergan et al.,
2007; Nesti et al., 2007).
However, before hESCs can be used for human cell therapy, regardless of their
differentiation potential and the functionality of their derived cells, they must be of
13
high quality to ensure genetic stability. They should also be derived under xeno-free
culture conditions (conditions free of non-human animal products, conditions
commonly referred to as animal-free) to eliminate the risk of transmission of animal
pathogens or immune reactions to animal proteins in human patients (Lei et al., 2007;
Unger et al., 2008).
Most hESC lines currently used in research were developed from cells
harvested from day 5 post-fertilization blastocyte-stage embryos donated from IVF
clinics, separated from the trophoblast and distributed over a culture plate previously
coated with a layer of mouse embryonic fibroblasts (MEFs) (Rodés et al., 2007). These
“feeder” cells are essential in releasing factors, particularly leukemia inhibitory factor
(LIF)—a growth factor that inhibits differentiation by encouraging the expression of
stem cell transcription factors which in turn maintain the expression of other
pluripotency genes—into the culture media (Mannello and Tonti, 2007; Mitalipova et
al., 2003). Although the media itself is synthetic and well defined, the exact cocktail of
factors released by the feeders cells is still poorly characterized and as of yet not fully
synthesizable, thus MEFS are still used by most research labs in the culture of ES cells,
be it human or not (Rodés et al., 2007). But MEFs may also shed non-human proteins
that have the potential to become incorporated into the hESC membranes and the
media itself often contains animal components such as fetal bovine serum (FBS) (Lei et
al., 2007; Unger et al., 2008). The donated embryos themselves are often of poor
quality (Unger et al., 2008; Van Royen et al. 1999). Good quality embryos have
14
blastomeres of roughly the same size with a single nucleus and very low accumulation
of anucleated fragments (Van Royen et al. 1999).
A number of animal-free ECM products are available for use as an alternative
to MEFs, including collagens, fibronectin, laminins, which have been used alone, in
combination, as 2D and 3D hydrogels or ECM scaffolds in attempts to mimic the
natural ECM from which the differentiated cell-type occurs in vivo (Freshney 2010;
Mallon et al., 2006). MEFs certified to be free of animal pathogens and human feeder
fibroblasts derived from newborn foreskin or adult marrow stroma cells have been used
in the development of clinical grade hESCs, but they tend to be more expensive and
more difficult to acquire than MEFs (Mallon et al., 2006; Skottmana et al., 2001). For
moral and ethical reasons use of human embryonic fibroblasts is not acceptable (Rodés
et al., 2007; Skottmana et al., 2001). Other alternatives to MEFS, such as the trademarked product Matrigel, are free of cells but are still derived from animal sources, and
often serve as a bridge between MEFs and complete xeno-free conditions, as they tend
to be well characterized and easy to use, but are still considered sub-optimal for clinical
purposes (Mallon et al., 2006; Skottmana et al., 2001). StemAdhere and CellStart are
other available xeno-free substrates available, but tend to be rather expensive. hESCs
grow well on mouse embryonic feeder cells; however, varying success has been
achieved with the available types of animal-free ECM products, which use media
supplemented with growth factors and signaling molecules that attempt to mimic the
factors supplied by MEFs, and thus are still less than ideal growth conditions that often
induce stress which not only can lead to spontaneous hESC differentiation, but also has
15
the potential to alter cellular properties (Bauer, 2011; Skottmana et al., 2001).
Numerous chromosomal abnormalities have been reported among hESCs cultured for
extended lengths of time on most of the available cell-free substrates (Bauer, 2011;
Skottmana et al., 2001).
To ensure the highest level of product quality, many nations, including the
United States, have outlined a stringent system of guidelines and laboratory techniques
(i.e. good manufacturing practices) for the procurement, derivation, treatment, and
storage of clinical-grade cell lines for potential therapeutic use (Crook, et al, 2007; Lei
et al., 2007). Recently, Crook and colleagues have successfully developed six human
embryonic cell lines that meet the requirements for classification as clinical-grade
human embryonic cell lines (Crook et al., 2007). These cell lines utilize human-derived
fibroblasts in GMP-manufactured bovine serum albumin replacement serum from high
quality embryos (Crook et al., 2007). The cell lines were karyotyped and carefully
screened for genetic stability and extensively tested for absence of pathogens. These
clinical-grade human embryonic stem cell lines are currently available to researchers.
In this study, clinical-grade hESC lines ESI-017 and EST-035 were evaluated
for their potential to differentiate towards hepatocytes in comparison to the results
obtained by Duan et al using the H9 line (2010). The lines ESI-017 and EST-035 were
grown on MEFs and the MEF-free culture substrate Matrigel, then differentiated
directly or transferred to collagen I coated plates prior to differentiation. Derived
hepatocyte-like cells derived characterized for hepatic markers, albumin secretion, and
ATP production, compared to that found by Duan et al (2010) and for primary
16
hepatocytes. Under these culture conditions, neither clinical-grade line was able to
produce derived-hepatocytes with the equivalent characteristics of primary hepatocytes
nor the stability or differentiation potential of the H9 line.
17
METHODS
Cell culture, expansion, and maintenance of cell lines.
hESC cell lines ESI-017 and ESC-035 were acquired from BioTime in a
cryopreserved state at passage 26 and 27, respectively. hESCs were recovered onto sixwell plates previously seeded with MEFs, in DMEM-F12 medium supplemented with
20% Knockout Serum Replacement (KOSR), 1% L-glutamine, 1% non-essential amino
acids, 2 ug/ml β-FGF, and 3.5 ul β-mercaptoethanol per 500 ml of media prepared.
hESCs were expanded for several passages on MEFs to stabilize the line before use in
experimentation. Passage occurred when the wells reached ~70-80% confluence.
Differentiating colonies were manually removed before each passage. Once colony
expansion occurred with minimal differentiation, hESCs were passaged onto Matrigel
(BD Biosciences) and maintained in mTeSR1 media (BD Biosciences).
iPSCs were acquired from WiCell at passage 33 in a cryoperserved state. They
were subsequently passaged by Dr. Ma of our lab for many additional passages,
cryoperserved and recovered as needed. iPSCs supplied for this project were of at least
passage sixty. iPSCs were maintained on MEFS in the above mentioned supplemented
DMEM-F12 medium and were acquired from Dr. Ma, as needed.
Adult human primary hepatocytes that have been freshly isolated from donor
livers were provided by the Liver Tissue Cell Distribution System of NIH and used in a
manner approved by IRB of the University of California, Davis, as described by Duan
et al. (2010). Primary hepatocytes were maintained in hepatocyte culture medium.
18
Induction of hESC and iPSC lines to definitive endoderm.
hESC cell line ESI-035, and iPSC line were each induced to definitive
endoderm (DE) while still on MEFs, under conditions without serum in RPMI 1640
medium supplemented with 100 ng/ml of Activin A, 2 nM L-glutamine, and 1%
antibiotic-antimycotic for 48 hours. For the following 3-6 days the medium was then
additionally supplemented with 1xB27 supplement and 0.5 mM sodium butyrate. hESC
cell line ESI-017, cultured primarily on Matrigel, without MEFs, was induced to DE
under the same conditions above with the addition of 25 ng/ml of Wnt3a during the
initial 48 hours. To explore the need for supplemental nutrients in the absence of
MEFS KOSR at 1% or 2% concentration was added during the first 48 hours.
Differentiation of hESC and iPSC-derived DE to hepatocytes.
If DE formed was of sufficient quantity, DE cells were split and reseeded onto
collagen I-coated plates at a suitable ratio, otherwise if DE formed was low,
differentiation towards a hepatic lineage was initiated directly, without further splitting
or removal off the Matrigel or MEFs. To differentiate the DE towards hepatocytes the
media was changed to IMDM medium supplemented with 20% FBS, 0.3 mM
monothioglycerol, 1% antibiotic-antimycotic, 0.126 U/ml human insulin, 100nM
dexamethasone, 20 ng/ml each of FGF4, HGF, BMP2 and BMP4, and 0.5% DMSO.
Media was changed every 48 hours for 14 days. After which differentiated cells were
further differentiated and maintained in HBM medium supplemented with the HCM
SingleQuots kit (Lonza), 1% antibiotic-antimycotic, 100nM dexamethasone, 20 ng/ml
each of FGF4 and HGF, 50 ng/ml oncostatin M., and 0.5% DMSO.
19
Detection of stage-specific markers through immunohistochemistry
fluorescence microscopy.
Immunohistochemistry (IHC) was used to detect the presence of stage-specific
markers characteristic of embryonic stem cells, definitive endoderm, mature as well as
immature hepatic lineage cells in determining the success of differentiation of stem
cells to derived hepatocytes. ESI-035 and ESI-017 hESCs were stained with antibody
for the pluripotency stem cell markers Oct4 (mouse antihuman; Milipore), Sox2 (rabbit
antihuman; Milipore), SSEA3 (rat antihuman; Milipore), and SSEA4 (mouse
antihuman; Milipore). ESI-035 and ESI-017-derived definitive endoderm was stained
with antibody for the presence of Sox17 (goat antihuman; R&D Systems) and FoxA2
(goat antihuman; R/D Systems). ESI-035 and ESI-017-derived hepatocyte-like cells
were stained with antibody for albumin (goat antihuman; Bethyl), alpha-feto protein
(rabbit antihuman; Sigma), and α1-antitrypsin (goat antihuman; Bethyl). iPSC-derived
hepatocyte-like cells and primary hepatocytes were stained with antibody to albumin
and human anti-trypsin.
Cells cultured in six-well plates were washed twice with PBS then fixed with
4% paraformaldehyde for twenty minutes at room temperature. The paraformaldehyde
was removed by washing the cells four times with PBS. Cells to be stained with
antibody were blocked and permeablized for two hours in blocking buffer consisting of
PBS, 0.3% Triton x-100 and 2% BSA, after which the blocking buffer was removed,
the appropriate antibody in 1 ml blocking buffer was added and the cells incubated
overnight at 4ºC. The following day, the cells were washed five times to remove the
20
primary antibody, and the cells were incubated for one hour secondary antibody in 1 ml
blocking buffer: Oct4, SSEA4 (Alexa 488 donkey antimouset); Sox2 (Alexa 594
donkey antirabbit); Alph-feto protein (Alexa 488 goat antirabbit), SSEA3 (Alexa 594
goat antirat); Sox17, Albumin (Alexa 488 donkey antigoat), FoxA2, α1-antitrypsin
(Alexa 594 goat antigoat) from Jackson ImmunoResearch. After one hour the cells
were washed five times with PBS then the PBS removed, 3 drops of mounting media
containing DAPI and a coverslip was applied and the cells were imaged using a Nikon
inverted fluorescent microscope.
Karyotyping of clinical-grade hESCs for genetic abnormalities.
Karyotying of clinical-grade hESCs was performed by Catherine Nacey of the
Dr. Jan Nolta lab at the UC Davis Medical center. Six-well plates of cells from the
ESI-035 cell line at passage 17 and passage 43 were provided. The cells of each sixwell plate were suspended at metaphase. 00 metaphases were examined. Twenty
metaphases were selected for closer analysis and three of the twenty were further
arranged on a karyogram.
ELISA quantification of albumin secretion by derived hepatocytes.
To determine the amount of albumin secreted by the ESI-017 and ESI-035
derived hepatocyte-like cells, media was retained at media change for every 48 hours,
and frozen in 96-well plates until use. For ESI-017 at passage 7 on collagen I, media
was recovered from day 16 to day 28 post-DE, for ESI-017 at passage 25 on Matrigel,
media was recovered from day 14 to day 22 post-DE, and for ESI-017 at passage 30 on
collagen I, media was recovered from day 6 to day 20 post-DE. Data points for ESI-
21
035 on MEFS at day 29, for ESI-035 on human feeders at day 29, and for ESI-035 on
Matrigel at day 21 were provided for comparison by Dr. Ma.
Amount of albumin present in samples was quantified using the Human
Albumin ELISA kit 488-120 (Bethyl Laboratories,Inc). Samples were processed
according to the manufacturer’s protocol. 96-well plates were washed five times with
wash solution. Wells were then incubated in affinity antibody at a concentration of
1:100 for one hour at room temperature, after which the plates were washed five times
with wash solution followed by 30 minutes incubation with blocking solution, washed
five times with wash solution, then 100 ul of samples, at a 1:10 dilution was added to
the appropriate wells. A standard curve was generated from a simultaneous plating of
standards of known albumin concentration ranging from 0 to 100 ng/ml. Plates were
incubated for 60 minutes then HRP detection antibody was added and incubated for
another 60 minutes, at which the plates were then read on a EMAX microplate-reader.
Cells from the wells in which media was recovered were tryponized to single cell
suspension in one ml culture media and counted on a hemacytometer. The
concentration of albumin in each sample was calculated from the standard curve then
converted to quantity of albumin secreted per one million cells.
Induction of P450 Cytochrome activity within derived hepatocytes.
To test for the presence and inducibility of the phase I enzyme p450 CYP2C9,
iPSC-derived hepatocyte-like cells at day 27 post-DE in a six-well plate were treated
with 25uM rifampicin (Sigma) and 0.5mM phenobarbitol (Sigma) in hepatocyte
maintenance media. Two wells were left untreated as negative controls and two wells
22
without cells, containing media only, were used for background. Media was changed
daily for 72 hours. The p450-Glo CYP2C9 Assay kit was used to assess induction.
Cells were processed as per the manufacturer’s protocol.
To test for the presence and inducibility of the phase I enzyme p450 CYP1A2,
ESI-017-derived hepatocyte-like cells at day 21 post-DE in a six-well plate were
treated with 25uM rifampicin (Sigma) and 100nM omeprazole (Sigma) in hepatocyte
maintenance media. Two wells were left untreated as negative controls and two wells
without cells, containing media only, were used for background. Media was changed
daily for 72 hours.
For each assay, the cells were washed, four times for the p450-Glo CYP1A2
and twice for the p450-Glo CYP2C9. Luminogenic substrate was added to each well
and incubated at 37ºC for 3 hours for p450-Glo CYP2C9 and 1 hour for p450-Glo
CYP1A2 then media was collected. Cells were rinsed with PBS, tryponized to single
cell suspension and counted on a hemacytometer. Equal volumes of media and
Luciferin detection solution was added to tubes and read on an EG&G Lumat LB 9507
luminometer.
Quantification of ATP produced by derived hepatocytes and primary
hepatocytes.
iPSC-dervied hepatocytes at days 14 and 28 post-DE and ESI-017-derived
hepatocyte-like cells at days 7 and 15, and primary hepatocytes were evaluated for
cellular ATP content using the ATP Assay kit (Invitrogen). Cells in six-well plates
were washed twice with PBS, treated with trypsin for ten minutes, separated to a
23
single-cell suspension by pippeting and collected into equal volumes of fetal bovine
serum (FBS), centrifuged for 5 minutes, resuspended in 1 ml culture medium and the
number of cells present counted on a hemacytometer. Cells were then lysed and the
supernatant collected as per the ATP Assay kit protocol. 100 ul aliquots consisting of
Assay buffer, reaction mix (prepared per assay protocol) and cell suspension quantities
ranging from 2ul to 40 ul were prepared and pipette onto a 96-well plate. Standards of
known ATP concentrations, prepared as per assay protocol were delivered to the same
plate. ATP content was detected by colorimeter and read on an EMAX microplatereader. A standard curve was generated and the concentration of ATP in each sample
calculated, first per ul of cell suspension then ATP per cell.
Evaluation of mitochondrial mass in derived hepatocytes by MitoTracker Green
FM staining.
To detect the presence and localization of mitochondria within iPSC-derived
hepatocytes and to assess the sensitivity of MitoTracker Green FM (Invitrogen), live
differentiated cells at day 24 post-DE of a six-well plate were washed once with PBS
then incubated with pre-warmed MitoTracker Green FM, prepared at 50nM and
100nM concentrations in hepatocyte maintenance media, for twenty minutes, after
which the MitoTracker Green was removed and replaced with PBS. Cells were then
imaged using a Nikon inverted fluorescent microscope.
24
Evaluation of mitochondrial mass of hESCs through derived hepatocytes by
Immunohistochemistry fluorescence microscopy.
Mitochondria Antibody (Rabbit antihuman, Milipore),which detects a 62 kDa
protein on the surface of human mitochondria, was used to detect the presence and
localization of mitochondria with iPSC-derived hepatocytes at day 24 and primary
hepatocytes at a concentration of 1:100 and 1:300, and ESI-017 hESCs,, ESI-017derived DE, and ESI-017-derived hepatocyte-like cells at days 6 and 15 post-DE at a
concentration of 1:100. Cells cultured in six-well plates were washed, fixed and treated
as previously described for immunoflourescence above, using Mitochondria Antibody
as primary antibody and donkey anti-rabbit secondary antibody (Alexa fluor 488) at a
concentration of 1:100 in 1 ml blocking buffer. Cells were imaged using a Nikon
inverted fluorescent microscope.
25
RESULTS
Culture, Induction, and Differentiation of iPSC line to derived Hepatocytes.
Vials of cryopreserved H9 hESCs were thawed and recovery was attempted by
Dr. Ma of our lab and Natalie from Dr. Nolta’s lab. Adherence was low and growth
slow on both MEFs and Matrigel. More often than not the cells failed to attached, died
or differentiated. iPSC cell line IMR90-4, was used in place of the H9 line as a control
for this project.
iPSCs on MEFs were supplied to me by Dr. Ma. Induction to DE was begun at
70% confluency. iPSCs formed colonies of compact cells, with distinct smooth borders
and high nuclear to cytoplassmic ratio, as expected of stem cells grown on MEFs
(Figure 1). Induction under no serum for 48 hrs then low serum, with the addition of
Activin A for 7-8 days produced a layer of cells covering 80% of the well with
definitive endoderm morphology: large triangular cells with clearly visible nuclei and
at least two distinct nucleoli and increased cytoplasm in comparison to that observed in
iPSC stem cell morphology (Figure 1).
DE derived from the iPSCs was allowed to differentiate towards hepatocytes
over 24-28 day, 15 days using the differentiation protocol and the remaining days in
hepatocyte maintenance media. Hepatocyte morphology began to emerge at day 9 postDE and by day 15 nearly all cells had a hepatocyte-like appearance. Cells had doubled
or tripled in size over the DE with a roughly hexagonal morphology. Double nuclei
were visible in many cells as well as abundant storage vesicles within the cytoplasm,
characteristic of hepatocytes (Figure 1).
26
A
B
C
Figure 1. Differentiation of iPSCs to derived hepatocytes. A) Colony of iPSC “stem
cells” growing on mouse embryonic feeder fibroblasts B) Definitive endoderm derived
from iPSC colonies, day 7. C) Hepatocyte-like cells derived from iPSC-derived
definitive endoderm, day 20.
27
Compared to the morphology of primary hepatocytes, that of the iPSC-derived
hepatocytes appears similar (Figure 2), although there were differences: derived
hepatocytes contained many more vesicles then the primary hepatocytes, and the nuclei
of the primary hepatocytes were more easily visible. Notable changes were observed in
the primary hepatocytes themselves over the three days period after arrival. Cell
borders became more visible as cells began to pull away from each other, vacuole size
and number decreased, patches of cells began to lift off the plate with media change.
iPSC-derived hepatocytes were then stained with MitoTracker Green FM in an
attempt to evaluate mitochondrial locatization. A concentration of 1:300 was found to
be toxic to the cells and they quickly detached from the plate. A concentration of 1:100
was tolerated, but also was too high and it was difficult to distinguish the mitochondria
network from background, although the network was visible in some of the cells, with
a concentration densest near the nucleus of the cell (Figure 2). MitoTracker Green stain
accentuated the hepatocyte-like morphology and presence of double nuclei in many of
the cells.
Immunohistochemistry (IHC) via fluorescence antibody staining for the
presence of albumin and the mature hepatocyte marker human alpha-1 antitrypsin
revealed a strong expression of both in most of the iPSC-derived hepatocytes, although
the primary hepatocytes still reflected higher expression with more intense stain and
presence of denser aggregates (Figure 3). The derived hepatocytes appeared larger than
the primary hepatocytes. Cultured cells spread out during differentiation.
28
A
B
C
Figure 2. Morphological comparison of iPSC-derived and primary hepatocytes. A)
iPSC-derived hepatocytes, day 20. B) Primary hepatocytes. 3 days after delivery C)
iPSC-derived hepatocytes stained with MitoTracker Green FM, 488
immunofluorescence, day 22.
29
A
B
C
D
Figure 3. IHC staining for hepatic markers of iPSC-derived and primary hepatocytes.
A) Albumin expression in iPSC-derived hepatocytes, day 20, B) Albumin expression in
primary hepatocytes, C) Human alpha-1 antitrypsin expression in iPSC-derived
hepatocytes, day 20 D) Human alpha-1 antitrypsin expression in primary hepatocytes.
30
Culture, Induction, and Differentiation of ESI-035 hESC line to derived
Hepatocytes on MEFs.
ESI-035 hESCs were acquired at passage 39 on MEFS and passaged. The hESC
colonies had crisp borders and compact stem cell morphology. IHC staining revealed
the expression of the stem cell pluripotency markers Oct4, Sox2, SSEA3 and SSEA4 in
all colonies (Figure 4). At 70-80% confluency the hESCs were induced to DE in a
manner similar to that for the iPSCs above. During induction to DE, more than 50% of
the hESCs died. By day 7, the majority of remaining cells demonstrated DE
morphology, although confluency was not more than 50-60%. Staining of two wells of
the DE cells revealed strong expression for the DE markers Sox17 and FoxA2 (Figure
5). Differentiation under the same conditions as the iPCS above resulted in a
population of cells displaying hepatocyte-like morphology, double nuclei and
hexagonal shape, but unlike the iPSC-derived hepatocytes there was minimal presence
of vesicles. Staining at day 16 post-DE for hepatocyte markers revealed a notable
amount of alpha-fetoprotein still present, production of albumin in about half the cells,
and most cells displaying some level of human alpha-1 antitrypsin, with a few
displaying intense punctuate aggregates, which were not seen in the iPSC-derived
hepatocytes (Figure 6).
31
A
OCT4
B
SOX2
C
SSEA3
D
SSEA4
E
Figure 4. ESI-035 hESCs on MEFS. A) ESI-035 hESC colony on MEFs. B-E) IHC for:
Oct4, Sox2, SSEA3, SSEA4.
32
A
SOX17
B
FOXA2
C
Figure 5. Induction of ESI-035 to definitive endoderm. A) ESI-035 DE on MEFs, B)
IHC for Sox17, C) IHC for FoxA2.
33
A
B
Albumin
D
C
Alpha-fetoprotein
Human alpha-antitrypsin
Figure 6. IHC staining of ESI-035 derived hepatocytes for hepatic markers. A) ESI-035
derived hepatocytes 20X objective, B) IHC staining for albumin 10X, C) IHC staining
for alpha fetoprotein 10X, D) IHC staining for human alpha antitrypsin 20X
34
Around day 18 post-DE occasional pockets of EMT (epithelial-mesenchymaltransition) was seen among the differentiating cells in some wells. EMT consists of
cells that have reverted back to a more fibroblast-like form and often appear motile.
EMT has been document in the culture and derivation of many cell types, including
hepatic lineage. EMT was not observed in the differentiation of iPSCs, but was
reported to occur in the differentiation of H9 to hepatocytes at around day 25.
Culture, Induction, and Differentiation of ESI-035 hESC line to derived
Hepatocytes on Matrigel.
ESI-035 hESCs were obtained at passage 37 on Matrigel. Colony borders were
not as crisp as colonies on MEFs, but this is to be expected, since it is the pushing back
on the hESCs by MEFS that contributes to the smooth borders. The cells are compact
with high nuclear-to-cytoplasmic ratio. Cells were expanded over two more passage
without incidence. Then at passage 39, the cells began showing poor adherence to the
plate after splitting and passage, colonies remained small and grew slowly, taking 10
days (5-6 days had previously been observed) to reach split confluency. Low adherence
coincided with a contamination issue in lab incubators and it was suspected that the
culture too was contaminated, but my lab manager decided against purchase of a kit for
detection of microorganism contamination, particularly mycoplasma, which is one of
the most common cell culture contaminants.
The colonies continued to grow slowly for two more passages. Then suddenly
at passage 41, there was high adherence and return to normal growth time, although
colony morphology had become increasingly spiky along the edges. In subsequent
35
passage holes appeared at the center of colonies where cells had died. IHC staining of
the hESCs for OCT4, SOX2, and SSEA3 appeared to be normal. But induction to DE
produced patches of both dense and low-confluency cells with varying morphology.
Many had the triangular DE form, but there were also clumps of undifferentiated cells,
and cells with spindle form and cytoplasmic projects. IHC staining for Sox17 showed
only about half the cells were expressing the marker (Figure 7).
Differentiation
towards derived hepatocytes produced cells that, by day 15, little resembled
hepatocytes and IHC staining for the hepatocyte markers, albumin, alpha fetoprotein
and human alpha-1 antitrypsin, revealed only very small patches of cells were positive
for either of the markers, particularly albumin.
Apparently the cells had lost the ability to properly differentiate and mutation
was suspected. It was also revealed that the passage initially obtained was not passage
37, but much older. The line had been obtained from BioTime at passage 26, stabilized
on MEFs after recovery from cryo storage for several passages then the passage
numbering reset to one when transferred to Matrigel. The initial passage acquired was
actually beyond passage 70.
36
A
B
SOX17
D
C
Albumin
E
Alpha-fetoprotein
F
Human alpha-antitrypsin
Figure 7. DE induction and hepatocyte differentiation of ESI-035 hESCs on Matrigel,
passage 41+. A) Low density area of induced DE showing abnormal cell morphology,
B) Denser area of induced DE, showing light expression of SOX17, C) 15 days postDE, cells showing little hepatocyte morphology, D) ICH staining for albumin, E) IHC
staining for alpha-fetoprotein, F) IHC for human alpha-antitrypsin. Circled inserts
show the location of expression of hepatocyte markers in the fixed well.
37
Karyotyping of late passage ESI-035 hESCs.
It is commonly reported in the literature that embryonic stem cells cultured past
passage 50 should be karyotyped for genetic integrity. After the revelation that the ESI035 cells were well beyond passage 70, not the passage 37+ as indicated on the plates
provided for this project, and considering the shift from low adherence to sudden
recovery and failure to differentiate properly, a plate of ESI-035 hESCs at passage “43”
was sent out of house to be karyotyped. Karyotying was performed by Catherine Nacey
of the Dr. Nolta lab. Cells were arrested at metaphase and 100 metaphases were
collected. 20 metaphases were further examined. 8 of the 20 (40%) of the metaphases
were found to contain an “iso-X” of the q arm (Figure 8). Meaning, one of the X
chromosomes had not separated from its sister chromatid during metaphase correctly.
Instead, the separation had produced an X chromosome containing both copies of the q
arm from both sister chromatids. No metaphases containing only p arms were found.
Such a genetic makeup could possibly not have been conducive to survival. Possible
deletions on the p arms of chromosomes 13 and 19, was also suspected, but Catherine
Nacey reported that these short arms are known for length variation in the normal
human population and could not say with certainty if there indeed were mutation. Dr.
Ma followed up with the kayotyping of an earlier passage of ESI-017, passage “17”
(actually around passage 45). The iso-X was not present in any of the metaphases
examined.
38
Deletion
Iso X
Figure 8. Karyogram of ESI-017 hESCs at passage “43”. Karyogram of ESI-017
hESCs at passage “43”, demonstrating an instance of iso-X of the q arm of an X
chromosomes (red arrow) and possible deletion of the p arm of one of the chromosome
13 pair (green arrow).
39
Culture, Induction, and Differentiation of ESI-0175 hESC line to derived
Hepatocytes.
ESI-017 hESCs were acquired at passage 25 on Matrigel. The hESCs had the
typical small, compact cell, high-nuclear-to-cytoplasmic ratio of embryonic stem cell
colonies. IHC staining for the pluripotency markers Oct4, Sox2, SSEA3, and SSEA4
reflected equivalent expression of all four markers as previously seen in iPSCs and
ESI-035 hESC on MEFs (data not shown).
Because low DE had been generated previously for the ESI-035 line, the initial
48-hour treatment to induce ESI-017 hESCs to DE was modified. The three treatment
groups explored produced significantly different results: 1% KSR+Activin A resulted
in spindly cells that were dead by day 5 of DE induction, 1% KSR+Activin A+Wnt3a
produced cells with more DE-like morphology by day 5 but the amount of DE was still
low. Treatment with Activin A+Wnt3a without KSR resulted in high-confluency of
cells with excellent DE morphology (Figure 9). This last treatment group was selected
for use with all subsequent ESI-017 hESCs DE induction. Hepatocyte-like cells
derived under this modified protocol demonstrated morphology equivalent to that seen
in the iPSC-derived hepatocytes, including double nuclei and the presence of an
abundance of cytoplasmic vesicles. Due to the large numbers of cells needed for the
ELISA, ATP, and CYP assays, these cells were designated for the aforementioned
assays and not stained for mature hepatocyte markers until passage 32 (see
Reassessment of ESI-017 by IHC, below). No formation of EMT was observed during
the differentiation of the ESI-017 line.
40
A
D
B
C
E
Figure 9. Induction to DE and hepatocyte differentiation of ESI-017. A) 24-hr trament
with 1%KSR+Activin A. B) 24-hr trament with 1%KSR+Activin A+Wnt3a. C) 24-hr
treatment with Activin A+ Wnt3a without KSR. D) DE at day 7. E) Derived
hepatocytes at day 16 post-DE.
41
Evaluation of albumin secreted per million cells by ELISA.
ESI-017 derived hepatocytes, differentiated on Matrigel or collagen I coated
plates were evaluated for secreted albumin over the post-DE date ranges indicated in
Figure 10. The results of these assays were a collaborative effort between two lab
members and myself, due to the amount of samples to be processed and time involved.
The greatest amount of secreted albumin was seen at day 22 for passage 25 cells on
Matrigel, an amount considerably greater than even the much younger passage 7 cells
on collagen I. Media was collected up to day 20 for passage 30 cells on collagen, but
only media up to day16 was usable as there was considerable debris in the media post
day 16 due to cells detaching from the plate. Evidence of detachment was seen at day
15 and by day 20 most of the cells had detached. Cell counts for the passage 30 assay
could only be estimated. As can be seen in Figure 10, passage 30 returned the lowest
amount of secreted albumin.
Primary hepatocytes were reported in Duan et al. to secret 100 ul albumin per
one million cells over 48 hours (Duan et al., 2010). All of the tested ESI-017
conditions measured in the nanogram range, with the exception of passage 25 at day
22, which at reported approximately 1.38 ug of albumin secreted over 48 hours per
million cells. In comparison to the primarily hepatocytes, the amount was still minimal.
A sampling of previous assays performed by Dr. Ma for early passages of ESI-035,
revealed that these lines too underperformed: day 24 on MEFs reported 10.5 ± 1.6 ug
of albumin secreted per one million cells, day 29 on human feeders reported 9.9 ± 0.8
42
Figure 10. Amount of Albumin secretion by ESI-017-derived hepatocytes. Amount of
albumin secreted per million cells per 48 hrs by ESI-derived hepatocytes over day
range post-DE from cells differentiating on Matrigel or collagen I coated plates. Media
was collected from day 6 to 16 for ESI-017 passage 30 (blue), from day 16 28 16 for
ESI-017 passage 7 (red), and from day 14 to 22 for ESI-017 passage 25 (green).
.
.
43
ug of albumin secreted per by one million cells, and day 21 5.3 ± 0.57 ug of albumin
secreted by per one million cells per 48 hours. The results of her assays reported
significantly more albumin secreted than the ESI-017, but was still no more than a
tenth of that reported for primary hepatocytes.
Cytochrome P450 induction assay of iPSC- and ESI-017-derived hepatocytes.
iPSC- and ESI-017-derived hepatocytes were first evaluated for the induction
of the cytochrome P450 isoform CYP2C9, an important drug metabolizing phase I
enzyme of hepatocytes, by 72-hour incubation with rifampicin, a chemical
demonstrated to increase the CYP2C9 activity in primary hepatocytes (33). Results
from the assay demonstrate that a greater amount of CYP2C9 was present in the more
mature iPSC-derived hepatocytes day 27 and ESI-017-derived hepatocytes day 22)
than in the ESI-017-derived hepatocytes at day 16, but that not much change in enzyme
production was seen between the control and rifampicin treatments of any of the cell
groups (Figure 11). Primary hepatocytes were not available at time of assay for
comparison.
Evaluation of the induction of the cytochrome P450 isoform CYP1A2, another
important drug metabolizing phase I enzyme of hepatocytes, was initiated with 72-hour
treatment with rifampicin and omeprazole in ESI-017-derived hepatocytes at day 15
post-DE and primary hepatocytes, but processing could not be completed due to the
derive-hepatocyte cells detached from the plate during washing prior to addition of the
substrate agent.
44
Figure 11. CYP2C9 rifampicin induction assay of iPSC- and ESI-017-derived
hepatocytes.
45
Evaluation of ATP production in iPSC-derived and ESI-017-derived
hepatocytes compared to primary hepatocytes.
The assay was performed with and without the consideration of glycerol
phosphate (Figure 12). Glycerol phosphate is a by-product of glycolysis, a process that
produces only a small amount of ATP through the breakdown of glucose that occurs in
the cytosol of a cell, without the assistance of mitochondria. According to the ATP
assays’ manufacturer, the presence of glycerol phosphate in a sample can give a falsepositive. When glycerol phosphate was not considered, the reported ATP from all the
derived hepatocytes appears similar to that reported from primary hepatocytes, with the
iPSCs performing better then the ESI-017s, which came in slightly lower than the
primary hepatocytes. An increasing trend in ATP present was observed with the iPSCderived hepatocytes from day 22 to day 24 post DE, which was expected of maturing
cells. However, the trend for the ESI-017-derived hepatocytes appears to be decreasing
with increasing day post-DE. Evaluation of the ESI-017 derived hepatocytes at day 15
was conducted but accidental over dilution of the cell extract produced results that
were far below the standard curve and not usable. With the loss of this data point we
were unable to confirm the decreasing pattern seen with the other two ESI-017 assays.
Since hepatocytes are very metabolically active cells, with large numbers of
mitochondrial to meet their huge energy demand, we decided there was a good chance
of significant amounts of glycerol phosphate production. When glycerol phosphate was
factored into the reported ATP produced by the cells, a large drop in ATP present was
46
Figure 12. Levels of ATP present in cell extract from iPSC and ESI-017- derived
hepatocytes. Measured at various days post-DE, and compared to primary hepatocytes,
with and without the consideration of the presence of glycerol phosphate. * assay
failed. ** assay not performed
47
seen in all the derived hepatocytes (red bars), but a huge drop was seen in the
primary hepatocytes, which was unexpected. Whether this measure is accurate for
primary hepatocytes or not is difficult to say. Review of the literature found that most
measure of ATP in primary hepatocytes was done using isolated mitochondria,
measured over a continuum and reported in nanomoles of ATP synthesized per minute
per milligram of mitochondrial proteins (Manfredi et al., 2002). Also, not considered is
that the primary hepatocytes were used immediately after arrival from overnight
shipment, from which they were sealed in leak-proof packaging and an air-tight
container on dry ice. Thus, the primary hepatocytes were metabolically slowed and in a
hypoxic environment, which is conducive to ATP production by glycolysis, not
oxidative phosphorylation. Once glycerol phosphate was subtracted from the assays, he
amount of ATP produced by both iPSC and line 017 derived hepatocytes was
remarkably close.
Because of the large amounts of cells needed to perform each assay at each cell
line time point, there were only enough cells to perform the assay at each time point
once. Several samples per assay were used to generate the average ATP produced per
cell. The error bars represent the deviation between the replicates on the same assay
and not between replicate assays.
Evaluation of mitochondrial mass by Mitochondrial antibody IHC.
MitoTracker Green FM requires live cells for use. Large numbers of cells
required for the above assays quickly depleted the supply of available cells. It was
decided to evaluate mitochondria present at the various differentiation stages and time
48
points using a mitochondrial antibody, that supposedly binds to a 62 kDa protein on the
surface of mitochondria, using a well from plates set aside for marker IHC, at the
suggested concentration range recommended by the manufacturer. Cells from hESCs,
DE, differentiating cells to derived hepatocytes, and primary hepatocytes were treated.
Unfortunately the antibody produced no signal in the 488 nm range of the secondary
antibody used to detect the primary mitochondrial antibody. It is known that the treated
cells were properly fixed and permeablized, since many of the marker stains were
performed at the same time and produced appropriate signal. As the staining was
performed at the end of the internship, there was not enough time or live cells beyond
the hESC stage available to reevaluate using MitoTracker Green FM.
Reassessment of ESI-017 by IHC.
With increasing passage of line ESI-017 on Matrigel, the line too began to
show signs, similar to the ESI-035, of irregular induction to DE. By passage 31 the
amount of cells that survived induction to DE steadily decreased and began taking on
an irregular morphology with cellular projections and deviations from the enlarged
triangular form. Spindles were increasingly present as well as becoming more loosely
associated with each other. IHC staining of passage 32 hESCs revealed good
expression of both Oct4 and Sox2 pluripotency markers, but low expression of SSEA3,
and no expression of SSEA4, which it was determined after the fact that the SSEA4
primary antibody was expired and most likely no longer viable
Staining of DE cells derived from passage 30 revealed only about half the cells
present were expressing the DE markers Sox17 and FoxA2. Attempts were made to
49
stain post-DE cells, but, as was experienced during the CYP and albumin ELISA
assays, the cells lifted from the plate during washing, passage 30 cells at day 15 postDE and even earlier, at day 9 for passage 31. What cells managed to be fixed and
stained showed no evidence of albumin production or human alpha-1 antitrypsin
expression, and only a minor amount of alpha-fetoprotein expression. As these cells are
in actuality beyond passage 60, considering the 27 passages by BioTime, the handful of
passages to stabilize the line from cryo storage and passage on Matrigel, they should be
karyotyped before further use, but not enough time remained of the internship to follow
up with the actual karyotyping.
50
DISCUSSION
At first glance it appears neither ESI-035 nor ESI-017 clinical-grade hESC line
could perform reliably and comparatively to iPSCs or H9 for differentiation towards a
hepatic lineage. The lines seem to be marked by genetic instability at late passage,
early formation of EMT, lower metabolic activity, and poor expression of mature
hepatocyte markers, compared to H9 and primary hepatocytes. But more thoughtful
consideration of the experimental protocol suggests that the culture environment may
play just as an important role in the differentiation potential of these cell lines as the
characteristics of the cell lines themselves.
Evaluation of the differentiation and derivation potential towards a hepatic
lineage of clinical-grade hESC cell lines ESI-017 and ESI-035 were to be based on the
results obtained by Duan et al., which employed the human embryonic cell line H9
(Duan et al., 2010). Differentiation of the H9 line was to serve as a control as the line is
stable and its ability to differentiate into hepatocyte-like cells well documented (Duan
et al., 2010). But at the time of this project the H9 cell line was not available. Both Dr.
Zern’s lab and Dr. Nolta’s lab, with whom we were collaborating, were having
difficulty recovering the H9 line from cryo storage. It was claimed by members of the
lab that iPSC cell line IMR90-4, acquired from WiCell was found prior to this project
to demonstrate an equal ability to differentiate into hepatocyte-like cells comparable to
the H9 line, although no data was r provided. It was used in place of the H9 line as a
control and differentiated along with the ESI lines under the same protocol, though
51
without any prior data presented to this project to the fact, it could only be assumed the
iPSCs performed equivalently to the H9.
In vivo, hepatocytes exist in a complex environment of collagen and fibronectin
ECM, under the influence of a constantly changing milieu of growth factors and
signaling molecules circulating in the bloodstream and released from neighboring nonparenchymal cells. Cell culturing protocol have not yet been able to successfully
replicate this environment. Growth on MEFs currently offers the most ideal system, as
MEFs appear to be able to supply an undefined cocktail of factors necessary to
maintain the stemness of hESCs. Matrigel, on the other hand, although derived from
mouse products must be supplemented with, to the best of our knowledge, the factors
necessary. The results of this project appear to demonstrate the inferiority of the
Matrigel system over MEFs. Only in the initial phase of this project were either
clinical-grade line cultured on MEFs, the remainder of the time Matrigel was used. The
H9s used by Duan et al. (2010) were maintained exclusively on MEFs. While it is
desired for clinical application to move from MEFs to a feeder-free culturing system,
the use of an alternate adherence substrate from that used for the H9, under the
influence of a different molecular cocktail, limits the comparability of the performance
of H9 and ESI cell lines. Use of the iPSCs as a control in this project suffered similar
limitations, as they too were exclusively maintained on MEFs and never evaluated on
Matrigel. But this project did demonstrate that these cell lines could proliferate and
differentiate into hepatocyte-like cells under feeder-free conditions. Although their
expression of hepatic markers appears to be lower than either primary hepatocytes or
52
iPSC-derived hepatocytes, as well as those derived from H9 by Duan et al. (2010), it
may simply be that the media cocktail needs to be further optimized to allow the cells
to reach their full potential as functional hepatocytes.
Passage number can also influence a stem cell’s ability to properly differentiate.
While in theory stem cells have the characteristic of endless renewal, in practice
genetic instability is often seen with increasing passage number due to accumulation of
random mutations. Not only age, but also stress on a cell due to exposure to a lessthan-ideal environment, chemicals, lack of nutrients, hypoxia, can induce mutation. For
this reason it is generally accepted that stem cells be karyotyed at the arbitrarily agreed
upon passage 50. Initially it appeared relatively low-passage clinical-grade cells were
being used for this project (between passage 25-38). But, it was revealed, as describe in
the results above, that none of the cells were at any passage younger than 60, and they
had not been karyotyed since use in our lab. Although younger passages existed in cryo
storage, these were designated for the lab’s more primary efforts defined by the focus
of their CIRM grant. The result of the late passage became evident in the ESI-035
line’s iso-X mutation, and mutation was possibly also responsible for the deceasing
performance of the ESI-017 line, which due to bounds of time of this project did not
allow for inclusion of follow up karyotyping. Apparently other labs have also
experienced increased incidence of mutation with long-term maintenance of hESCs on
the Matrigel system, again pointing to its less-than-ideal culturing ability (Bauer,
2011). Passage number of the H9 line was not reported in the Duan et al paper (2010).
It may be too that H9s suffer the same problem with late passage, but the H9 line has
53
been used by many labs for several years. More than likely the H9 is a stable line, well
beyond passage 50, and that these new clinical-grade lines do not share the same
degree of stability.
This project was plagued by poor cell growth, low production of DE induction
and directed differentiation as cells aged, high cell death, and spontaneous
differentiation and due to the constant battle to produce derived-hepatocytes from these
late passage hESCs with good hepatocyte marker expression, it was difficult
maintaining sufficient supplies of cells for the ELISA and cytochrome P450 assays, as
these assays often required an entire six-well plate of cells per assay. Part of this was
alleviated by pooling resources with other team members, not only to share the work
load, as some assays were time consuming, time sensitive, and required processing of
many samples, but it allowed us to share data from a wider sampling of cells and
passage numbers. Ideally, the lower passage cells should have been expanded
horizontally, that is when a plate was ready to split, each well should have been split
onto six additional plates. Instead, cells were passaged vertically; one or two wells
would be used to maintain the hESC line while the rest of the plate was induced
towards DE. This resulted in cells from varying passages used for each assay. In part,
this was due to the expense of the growth factors necessary for DE induction and
hepatocyte differentiation, particularly Activin A and especially Wnt3a. But horizontal
passage would only be useful for laboratory studies. To be useful for clinical purposes
vertical passage is actually more important as vast number of cells would be needed for
patient treatment, which would call for many rounds of passage. It would have been
54
interesting to evaluate these lines at the same passage, but it indeed these cells cannot
show the stability with increasing passage as the H9, which appears to be the case
based on the increasing difficulty generating good DE with increasing passage number,
there is no need to conduct functional assays with them.
To make matters worse, this project suffered from constant direction change.
Despite my best efforts to chart out a course to best utilize the cells available and to
ensure a continued supply of cells, actual practice was at the whim of my lab manager
who continually shifted the focus from day to day. Further, early induction of ESI-017
hESCs produced excellent DE at lower passage, which, due to deadlines in other lab
projects, the plates, initially designated for assay of this project, were often
commandeered for use elsewhere, leaving fewer cells available for this project. But on
the flip side, if the quality of my DE cells, particularly the ESI-017-derived DE was
sufficient to attract the attention of others and it is indeed the culturing conditions that
need to be optimized, once achieved to remove the vertical passage problem, these
cells could have the potential to become a cell product other outside labs might want to
use, particularly drug companies looking for hepatocytes for a more accessible source
than the limited supply from human sources currently available
This project demonstrated that cell lines derived from these blastocyts do not
necessarily have equivocal potential for research and clinical use and the ability to
differentiate into specific lineages. While H9 demonstrate the ability to give rise to
hepatocyte-like cells, albumin and metabolic, studies by Duan et al. and others have
shown that they are not equivocal to primary hepatocytes (Duan et al, 2010). Variation
55
too was seen in the potential for the two ESI lines evaluated in this study, not only
from each other but from H9 and iPSC-derived hepatocytes as well. The ESI-035 line
showed evidence of EMT earlier than the H9, while the ESI-017 line never manifested
it. It appears that, as indicated by Dr. Ma’s ELISA results, derived hepatocytes from
the ESI-035 line could potentially produce albumin equivalent to that of the H9 if the
passage number is low and grown on MEFs, but the ESI-017 line could barely produce
a tenth of that of the ESI-035 line, and neither line was capable of matching the H9 line
when grown on Matrigel. Both lines may have genetic stability issues at late passage,
but whether this is an artifact of culture on Matrigel or intrinsic to the lines themselves
needs to be explored further. The ESI-017 line appears to be a better choice if one
wants to avoid the EMT issue, but line ESI-035 could potentially hold more promise
for hepatocyte differentiation if the formation of EMT is closely monitored.
Cell availability limited the number of cytochrome, albumin, and ATP assays
that could be performed. Some of the assays performed in this project were not
performed in the Duan et al. (2010) study with the H9, particularly the mitochondrial
evaluations. Mitochondrial staining with MitoTracker products are a well-established
protocol that, once the optimized concentration and incubation time is determined for a
particular cell type, produce excellent results in revealing mitochondrial localization
and relative mitochondrial mass. Use of MitoTracker, be it Green FM or any of the
other MitoTracker variants, should have been employed for the above mentioned assay
instead of the mitochondrial antibody that produced no results. The mitochondria
evaluations in this study had been aimed mostly at mature derived hepatocytes, but
56
lines, including iPSCs and H9, should also have been evaluated throughout the
differentiation process to give a clearer picture of the progression of mitochondria
expansion and to confirm that the ESI hESC lines do indeed have the lowmitochondria, perinuclear characteristic found in other hESC lines, since it has been
documented in the literature that loss of perinuclear localization has been found to
coincide with late passage and loss of pluripotency. But since MitoTracker Green FM
was abandoned and the mitochondria antibody failed to produce any results no
conclusions can be made from this study as to the equivalence of mitochondria mass in
ESI-derived hepatocytes and primary hepatocytes or the H9 line.
Part of the intent of this project was to explore mitochondrial mass and
functionality, as one possible avenue to explain the difference in albumin production
between derived hepatocytes and primary hepatocytes. ATP assays were conducted
using cells at whatever day post-DE that happened to be available. The iPSCs were
meant to serve as a control, yet ATP was not measured in either ESI line at the same
day points as the iPSCs. Better matched controls need to be considered and more than
one replica for each day point needs to be conducted for statistical significance.
Comparison to previous studies was difficult to do, since most published work
measured ATP production over a continuum and calculated nanomoles ATP synthesize
per time period per amount of mitochondria, not at single moments in time. It is not
absolutely necessary to measure ATP in the manner that has been done in previous
studies, since part of the goal of this project was to evaluate whether ATP production
may play a role in albumin secretion levels, which were also measured at specific time
57
points. But to be of value in answering the albumin question, the ATP assay time
points would be of more value if they matched the time points for albumin secretion.
Only a small number of ATP assays were able to be performed, due mainly to the
number of assay reagents needed for the six-well plates. Not enough time points were
assayed in this study to be able to draw any conclusions linking albumin secretion and
ATP production. From the assays that were done, it appears the ESI-017 line
demonstrates the ability to produce a significant amount of ATP comparable to that
found in the iPSCs once glycerol phosphate was and that a majority of it is being
produced via oxidative phosphorylation, indicative of good mitochondrial function, but
more assays are need to refine the true proportions and reduce the error generated in
this study.
The initial design of this project was to include primary hepatocytes at each
stage of analysis alongside the derived hepatocytes, but it quickly became apparent that
primary hepatocytes are difficult to acquire, as when they become available from
outside sources, there are far more researchers requesting them then are available. This
makes it all that much more imperative that when they do become available, that they
be allowed to recover properly prior to use but also used in a timely manner. The ATP
assay conducted during this project including the primary hepatocytes indicated a high
amount of glycerol phosphate had been present. This could have been an artifact, but it
could also mean the primary hepatocytes had not had enough time to recover from a
hypoxic condition to one more conducive to oxidative phosphorylation and efficient
production of ATP. Age of primary hepatocytes may also have played a role in other
58
evaluations in this study. Morphological photographs were not taken until three days
after the arrival of the primary hepatocytes to the lab. Obvious changes in cell-cell
adhesion and vesicle content was observed over this period, indicating the primary
hepatocytes were beginning to lose their hepatic functionality and dying.
Only after equivocal evaluation of the ESI lines to H9 on MEFs, and possibly
primary hepatocytes, has been undertaken, and the performance of each line
characterized relative to each other, should the study have moved to feeder-free
conditions. Without characterization of the ESI lines on MEFS, in comparison to the
H9 line, the significance of the differences between the ESI lines on feeder-free
substrate and the H9 line cannot accurately be assessed. But if use of animal-free
conditions and clinical-grade hESCs are desired, it may be more appropriate to
continue refining the culture conditions for the ESI lines and comparing them to the
performance of primary hepatocytes rather than the H9 line, which is not clinicalgrade.
59
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