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Hepatic differentiation in human naïve stem cells compared to human embryonic stem cells

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Hepatic differentiation in human naïve stem cells compared to human embryonic stem cells

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Hepatic differentiation in human naïve stem cells compared to human
embryonic stem cells

By

Samantha Nguyen

A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(MOLECULAR MICROBIOLOGY AND IMMUNOLOGY)

August 2015

Copyright 2015 Samantha Nguyen

Acknowledgements
I would like to thank my family and friends for their help and support, especially to my
parents and my little brother. It is with their encouragement that I am able to make it this
far.
I would also like to thank Dr. Toshio Miki for his mentorship. I am grateful for the
guidance and learning opportunities given to me in his lab.
Last but not least, I would like to thank my former and current lab members for their help
and support: Irving Garcia, Natalie Rodriguez, Kevin Parducho, Ristin Drewry, Jennifer
Jung, Jennifer Izumi, Mai Hamano, Dr. Mohamed Hammad, Anthony Gonzalez,
Ludivina Vazquez, Lisa Yanuaria, Omar Lopez, and McMillan Ching.
i
Table of Contents
List of Figures .................................................................................................................. iii
List of Tables ................................................................................................................... iv
List of Abbreviations ........................................................................................................ v
Abstract ........................................................................................................................... vi
Chapter I: Introduction ..................................................................................................... 1
1.1 Liver Disease ......................................................................................................... 1
1.2 Human Pluripotent Stem Cells ............................................................................... 2
1.3 Naïve State Stem Cells .......................................................................................... 3
1.4 Differentiating Pluripotent Cells to Hepatocytes ..................................................... 5
Chapter II: Hypothesis ..................................................................................................... 7
Chapter III: Materials and Methods ................................................................................. 8
3.1 Cell Culture and Maintenance ................................................................................ 8
3.2 Fluorescent-acquiesced Cell Sorting .................................................................... 10
3.3 RNA Extraction of Hepatocyte-like Cells .............................................................. 10
3.4 Real Time quantitative PCR ................................................................................. 10
3.5 Urea Assay .......................................................................................................... 11
3.6 Albumin ELISA ..................................................................................................... 11
3.7 Periodic Acid Schiff Staining ................................................................................ 12
3.8 Statistical Analysis ............................................................................................... 12
ii
Chapter IV: Results ....................................................................................................... 13
4.1 Morphology .......................................................................................................... 13
4.2 FACs .................................................................................................................... 14
4.3 RT-qPCR ............................................................................................................. 15
4.4 Urea Assay .......................................................................................................... 16
4.5 Albumin ELISA ..................................................................................................... 17
4.6 PAS Staining ........................................................................................................ 18
Chapter V: Discussion and Conclusion ......................................................................... 19
References .................................................................................................................... 23

iii
List of Figures
Figure 1: Morphological change of human embryonic stem cells to the naïve state ....... 4
Figure 2: Hepatic lineage ................................................................................................ 5
Figure 3: Cell Morphology throughout hepatic differentiation ........................................ 13
Figure 4: Morphology of undifferentiated cell ................................................................ 14
Figure 5: Fluorescent-acquiesced cell sorting for asialoglycoprotein receptors ............ 14
Figure 6: Relative hepatic gene expressions in differentiated stem cells ...................... 15
Figure 7: Urea production in differentiated stem cells ................................................... 16
Figure 8: Albumin production in differentiated naïve stem cells and primed stem cells . 17
Figure 9: Glycogen storage in differentiated naïve stem cells and primed stem cells ... 18

iv
List of Tables
Table 1: Small molecules used for naïve stem cell media ............................................... 8
Table 2: List of targeted genes and their forward and reverse primers ......................... 11

v
List of Abbreviations
ESC= Embryonic Stem Cell
hESC= Human Embryonic Stem Cell
mESC= Mouse Embryonic Stem Cell
hPSC= Human Plurpipotent Stem Cell
LIF= Leukemia Inhibitory Factor
ICM= Inner Cell Mass
NHSM= Naïve Human State Media
H9-Hep= H9 Derived Hepatocyte-like Cells
H9 NHSM-Hep= Naive State H9 Derived Hepatocyte-like Cells

vi
Abstract
With the potential of using stem cells to provide a large source of hepatocytes for
liver modeling or cell therapy, human naïve stem cells are becoming the topic of many
studies. Past studies have divided stem cells to two states: naïve and primed. Since the
stem cells in mice are pluripotent and considered less advanced in the differentiation
process, they are considered in the naïve state. On the other hand, human embryonic
stem cells show characteristics of being in the primed state. Thus, studies have
reprogrammed human embryonic stem cells into a naïve state to reflect the mouse
embryonic stem cell-like characteristics. In our study, we induced hepatic differentiation
in both human embryonic stem cells and human naïve stem cells to compare their yield
of hepatocyte-like cell. Because of the mouse embryonic stem cell-like characteristics,
we hypothesized that the human naïve stem cells would efficiently differentiate to
hepatocyte-like cells compared to human embryonic stem cells. A modified standard
protocol was used to differentiate the cells and the resulting cells were checked for
hepatic characteristics. The results of this study showed that there were no significant
differences in the yield of hepatocyte-like cells. However, the differentiated human
embryonic stem cells had a tendency to be slightly more mature than the differentiated
human naïve stem cells. Further studies are needed to explore the cause for this and to
optimize hepatic differentiation in both cell types.

1
Chapter I
Introduction
1.1 Liver Disease
Liver disease affects one in 10 people- at least 30 million people- in the U.S.
According to the American Liver Foundation, liver disease consists of many different
types such as alcoholic liver disease, fatty liver disease, drug-induced hepatitis, viral
hepatitis, and liver cancer, etc. Liver disease can cause fibrosis and cirrhosis, which
eventually led to end stage/decompensated liver disease [2]. When liver disease has
reached the critical stage, liver transplantation is the only option of treatment.
Unfortunately, liver transplantation is limited due to organ shortage. Currently, about
16,000 patients are on the waiting list and only 123,392 liver transplants have been
performed in 25 years. This is an average of roughly 4,935 liver transplants per year.
However, around 29,424 patients on the waiting list have died while waiting for liver
transplants [1].
Due to the limitations of liver transplantation as a treatment, alternative therapies
are being explored. For example, cell therapy is an alternative currently being studied.
There are types of cell therapies categorized by the route of therapy and the cells
involved. For instance, there are extracorporeal or ex vivo therapies where bioartificial
livers are developed to support patients. Cell therapy can also be done in vivo where
cells are implanted into patients for liver repair. Both of these methods require cells that
possess hepatic functions. These can include isolated primary cells, progenitor cells,
and cell lines such as tumor-derived cells and immortalized hepatocytes. However,
2
primary hepatocytes are not readily available and other cell line cells are not fully
functional hepatic cells. Thus, generating functional hepatocytes with human ESCs for
cell therapy is a more attainable route [3].
1.2 Human Pluripotent Stem Cells
There are many different types of stem cells. However, what sets human
pluripotent stem cells (hPSC), including embryonic stem cells and induced pluripotent
stem cells, apart are that they can maintain their proliferation abilities and still keep their
pluripotency [4]. Embryonic stem cells (ESCs) are derived from the inner cell mass of
blastocysts [7]. They are capable of self-renewal and differentiating into lineages of all
three germ layers, both in vitro and in vivo. These three germ layers are the mesoderm,
ectoderm, and endoderm. In order to maintain ESCs in their undifferentiated state, they
need to be cultured in conditions with exogenous factors and feeder cells; irradiated
mouse embryonic fibroblasts (MEFs) [6].
Pluripotent cells can be categorized in either the naïve state or the primed state
[5]. The primed state is more advanced in the differentiation process whereas the naïve
state is considered to be at the much earlier stages. The term naïve state is
interchangeable with the term ground state [6].
Both mouse ESCs and human ESCs are derived from the ICM of blastocysts.
Although they were derived at the same stage of development derived from
preimplantation embryos, hESCs were significantly different from mESCs and similar to
mouse epiblast-derived stem cells. They showed different morphology and had different
mechanisms to control pluripotency. For example, mESCs in culture have dome shaped
3
colonies whereas hESCs tend to be more monolayered. Furthermore, mESCs require
leukemia inhibitory factor (LIF) to maintain their pluripotency whereas it is believed that
bFGF is needed to maintain the pluripotency of hESCs [6].
Instead, the hESCs’ morphology and epigenetics were more similar to mouse
epiblast stem cells derived from mouse embryos. Furthermore, mESCs are
characterized as having the ability to form chimeras when reintroduced to
preimplantation embryos. hESCs lack this ability [6]. In addition, the cell fate of hESCs
tends to be directed towards the ectoderm lineage, making them biased for
differentiation [17]. Furthermore, mESCs have a higher single-cell cloning efficiency
compared to hESCs. mESCs can survive single-cell passaging whereas hESCs have a
lower survival rate [8]. These observations lead to the suspicion that hESCs are already
at a more differentiated state compared to their murine counterpart [6]. Therefore,
mouse ESCs are considered to be in the naïve state, and human blastocysts are
considered to be in the primed state.
These differences led to the research on inducing hESCs to the naïve state [8].
1.3 Naive State Stem Cells
Stem cells with similar characteristics to mESCs, and are considered to be at a
much earlier developmental stage, are defined as ground state stem cells or naïve stem
cells. As mentioned earlier, the difference between mESCs and hESCs could be due to
the conditions the hESCs are cultured in. Thus, reprogramming hESCs into the naïve
state are being explored [6].
4
Several studies have tried reprogramming and maintaining hESCs from the
primed state to the naïve, ground state stem cells by culturing the cells in conditions that
contain various small molecules. One example is to reprogram hESCs to the naïve state
by enhancing STAT3 activity. The study looked for expressions of NANOG and OCT4
among other transcription factors to determine the induction to the naïve state [9].
In another study, hESCs are cultured in conditions that contained LIF, TGFb1,
and bFGF. Additionally, PD0325901, CHIR99021, SP600125, and SB203580 are
included in the culture conditions. These are ERK 1/2 inhibitors, GSK3b inhibitors, JNK
inhibitors, and p38 inhibitors respectively. Rock inhibitor and PKC inhibitors can be
added to the conditions, but they are optional. When the cells were cultured under this
naïve human stem cell condition (NHSM), it was observed that the morphology of the
cells became similar to that of the mESCs. The cell colonies became more dome-like
[8].

Furthermore, the human naïve stem cells derived from hESCs cultured in the NHSM
conditions were microinjected into mouse morulas and this lead to the generation of a
chimera. This may support the induction of hESCs to the naïve state [8].

Figure 1. hESCs
morphological change to
dome shaped colonies like
mESCs [8].
5
1.4 Differentiating Pluripotent Cells to Hepatocytes
Although naïve cells have been differentiated in vivo to the three different germ
layers, they have not yet been differentiated to fully functional hepatocytes both in vivo
and in vitro. On the other hand, many protocols have been produced to try and
differentiate stem cells to mature, functional hepatocytes in vitro.
Earlier protocols for hepatic differentiations have tried forming embryoid bodies
as the primary step. This method relies on spontaneous differentiation of the pluripotent
cell aggregates to a mixed population of the three different germ layers. Since then,
studies have tried to improve the efficiency of the differentiation steps to allow for higher
yield of hepatocyte-like cells [10].
As mentioned before, stem cells can be pushed to three different lineages:
endoderm, mesoderm, and ectoderm. With the focus on hepatocytes, they are derived
from the endoderm lineage. Thus, differentiation protocols will start with inducing stem
cells to the definitive endoderm stage which can be characterized by CXCR4,
chemokine receptor type 4, and SOX17, a transcription factor [11, 12].

Figure 2. Hepatic lineage
6
The number of stages of hepatic differentiation varies between protocols. One
protocol in particular divides the differentiation process into four different phases:
definitive endoderm, specified hepatic endoderm, immature hepatocytes, and mature
hepatocytes. Each stage requires culturing hESCs in conditions with different
recombinant growth factors [12]. Many protocols have similarities by using the same
growth factors such as activin A, hepatocyte growth factor (HGF), and oncostatin M
(OSM). The use of these growth factors has been based on developmental signaling. In
addition, many groups are looking at many other growth factors as possible candidates
for better differentiation to hepatocyte-like cells [10].
Obtaining stem cell-derived hepatocytes has potential in providing a large
resource of cells for liver disease studies as well as possible therapeutic effects. For
example, these stem cell-derived hepatocytes could lead to an alternative approach to
therapy instead of organ transplantation. Thus, increasing the efficiency and yield
hepatic differentiation is important. One way of increasing the yield of functional
hepatocyte-like cells derived from pluripotent cells can be through the use of naive stem
cells.

7
Chapter II
Hypothesis
Previous studies showed that human embryonic stem cells are not considered
naïve and could be primed to a specific lineage compared to mouse embryonic stem
cells. These propagated many more studies to reprogram human embryonic stem cells
into the naïve state with various methods. And as a result, these newly obtained human
naïve stem cells show more mouse ESC-like characteristics, which is not primed toward
a specific lineage.
Thus, we hypothesize that human naïve state stem cells can more efficiently
differentiate to hepatocyte-like cells compared to human embryonic stem cells. They
should have higher levels of expression of hepatic genes and increased hepatic
functions compared to human embryonic stem cell-derived cells.

8
Chapter III
Materials and Methods
3.1 Cell Culture and Maintenance
The human embryonic stem cells were obtained from the USC Stem Cell Core.
The H9 cells were cultured on 100 mm tissue culture plates coated with 1% matrigel in
mTeSR1 media (StemCell Technologies). For use in differentiation to hepatocyte-like
cells, the H9 cells were passaged to 6-well plates that were also coated with matrigel
and consisted of mTeSR1 w/rock inhibitor media.
The human naïve stem cells were acquired by culturing the human embryonic
stem cells under the NHSM conditions to induce the cells to ground state. The human
naïve stem cells were first cultured on mouse embryonic feeder cells in 60 mm tissue
culture plates.
Human LIF 20ng/ml
TGFb1 1ng/ml
bFGF 8ng/ml
PD0325901 1uM
CHIR99021 3uM
SP600125 10uM
SB203580 10uM
Y27632 (rock inhibitor) 5uM
Table 1. Small molecules used for NHSM conditions
9
For cell expansion, the cells were passaged to matrigel coated 100 mm tissue
culture plates in mTeSR1 media. Once enough cells were obtained, the ground state
stem cells were passaged from the 100 mm tissue culture plates to 6-well plates coated
with matrigel in mTeSR1 w/rock inhibitor.
Once the differentiation process began on both the H9 and H9 NHSM cells in the
6-well plates coated with matrigel, the culture conditions varied depending on the stages
of the differentiation process.
Induction to Hepatocyte-like cells
Day 1-Day 5 (Definitive Endoderm Stage)
Both H9 and H9 naïve stem cells were cultured on matrigel-coated plates under
conditions given by the Definitive Endoderm Kit from StemCell Technologies.
Day 6-10 (Specified Hepatic Endoderm)
The cells culture conditions were changed to RPMI/2% B27 supplemented with
20ng/ml BMP4 and 10ng/ml FGF2.
Day 11-15 (Immature Hepatocytes)
The cells were then cultured in RPMI/2% B27 supplemented with 20ng/ml HGF.
Day 16-20 (Hepatocyte-like cells)
The cells were then cultured in Clonetics Hepatocyte Culture Medium (HCM) with
‘Singlequots’ (supplied by kit) supplemented with 20 ng/ml of Oncostatin-M. EGF was
not added to the media.
10
3.2 Fluorescent-acquiesced Cell Sorting
By the end of the cell differentiation process, the cells were rinsed with PBS and
dissociated with accutase. FACS buffer was added to each well to dilute the accutase.
The cells were then resuspended in FACS buffer and incubated with ASGPR-FITC
antibodies (Hycult). Afterwards, the cells were washed and resuspended in FACS
buffer. The cells were sorted via ARIA II for expression of ASGPR receptors.
3.3 RNA Extraction of Hepatocyte-like Cells
RNA was extracted from both the H9 and H9 NHSM derived hepatocyte-like cells
by using Trizol-LS and the Qiagen micro-Rneasy kit. The protocol was adjusted by
using part of the protocol provided with Trizol-LS and the protocol provided by the
Qiagen Rneasy kit.
3.4 Real Time quantitative PCR
Total RNA was extracted from the hepatocyte-like cells. The entire total RNA was
used with qScript
TM
Reaction Mix and qScript
TM
Reverse Transcriptase (Quanta
BioSciences) for the real time RT PCR to synthesize cDNA. Quantitative real-time PCR
was done using PerfeCTa SYBR Green SuperMix- Low Rox (Quanta Biosciences) and
the forward and reverse specific primers for each gene of interest. The results were
normalized against GAPDH in hepatocytes.

11
Genes of Interest Forward Primer Reverse Primer
GAPDH TGC ACC ACC AAC
TGC TTA GC
GGC ATG GAC TGT
GGT CAT GAG
HNF4A AGC AAC GGA CAG
ATG TGT GA
TCA GAC CCT GAG
CCA CCT
Factor X AGA TTC AAG GTG
AGG GTA GGG
GAC CAC CTC CAC
CTC GTG
Albumin AAT GTT GCC AAG
CTG CTG A
CTT CCC TTC ATC
CCG AAG TT
Table 2. List of targeted genes including forward and reverse primers
3.5 Urea Assay
The differentiated cells were exposed to 0mM, 5mM, and 10mM NH
4
Cl. Cell
culture supernatant was collected at 1 hr. and 2 hr. of exposure. Urea production by the
differentiated cells was measured with the QuantiChrom Urea Assay kit (DIUR-500).
The final concentration of urea in the supernatant was measured using the colorimetric
assay and calculated per the kit’s instructions.
3.6 Albumin ELISA
Cell culture supernatant was collected at day 10 and day 20 of the differentiation
process. Albumin production was determined from a standard curve with the Albumin
ELISA quantitation set (Bethyl Laboratories, Inc.). First the assay plates were coated
with antibody provided by the kit for 1 hr. Then the plate was washed and incubated
with blocking solution for 30 minutes at room temperature. Then the plate was washed
12
and the samples were added to the wells and incubated for 1 hr at room temperature.
The plate was washed once again and incubated in diluted HRP detection antibody for
1 hr. Afterwards, the plate was washed and incubated with TMB substrate solution in
the dark at room temperature for 15 minutes. Stop solution provided by the kit was
added and the plate was measured on a plate reader at 450nm.
3.7 Periodic Acid Schiff Staining
Glycogen storage was determined in the hepatocyte-like cells. First, the cells
were fixed with 4% PFA. Then, PAS staining was performed by following the protocol
given in the PAS staining kit from Sigma Aldrich. The cells were incubated in Periodic
acid solution for 5 minutes at room temperature. Next, they were rinsed with distilled
water. Afterwards, the cells were incubated in Schiff’s reagent for 15 minutes at room
temperature before being washed in distilled water for 5 minutes. Lastly, they were
incubated in Hematoxylin Solution, Gill No. 3, for 90 seconds before washing in distilled
water and air dried.
3.8 Statistical Analysis
Results are expressed as mean ± standard error of mean (SEM). Groups were
compared with Student’s t-test and a value of p<0.05 was considered statistically
significant.

13
Chapter IV
Results
4.1 Morphology

Figure 3. Morphology of cells from day 0, 5, 15, and 20 of differentiation taken at 10x.
14

Figure 4. Morphology of cells from day 0 at 4x.

As shown above, Both H9-Hep and H9 NHSM-Hep cells undergo morphological
changes during differentiation. The cells have increased in size and become more
monolayered. Furthermore, it seems like the morphology of the H9-Hep cells at day 20
looked more polygonal like hepatocytes. The shape of the H9 NHSM-Hep cells at day
20 looked more like immature hepatocytes. Thus, it is possible that the H9 derived cells
yielded more mature hepatocyte-like cells during differentiation.
4.2 FACs

Figure 5. ASGPR presence in H9 derived cells and H9 NHSM derived cells

15
To quantify the yield of differentiated cells to the hepatic lineage, FACS was
performed to look for asialoglycoprotein receptor (ASGPR), a liver specific receptor, in
the differentiated cells. The results showed that H9-Hep cells were more ASGPR
positive than the H9 NHSM-Hep cells. However, both cell types had low expression for
the receptor overall. Furthermore, the difference between the H9-Hep cells and H9
NHSM-Hep cells were not significant. This could indicate that both cell types have not
yet fully differentiated to mature hepatocyte-like cells.
4.3 RT-qPCR

Figure 6. The gene expression of HNF4a, Factor X, and Albumin in H9-Hep and H9
NHSM-Hep cells relative to the gene expressions in hepatocytes. They have been
normalized against GAPDH.
16

The real time qPCR resulted in H9 NHSM-Hep cells having higher gene
expression in factor X and albumin than H9-Hep cells whereas H9-Hep cells had higher
gene expression in HNF4a. The difference in gene expressions between the two cell
types was not significant. As a result, it is possible that differentiating H9 and H9 NHSM
to the hepatocytes may not have different yields. In addition, the gene expression levels
were low compared to hepatocytes.
4.4 Urea Assay
H9-Hep
H9 NHSM-Hep
Hepatocytes

Figure 7. Urea production by the differentiated cells and hepatocytes after 2 hours of
exposure to ammonia at 0mM and 5mM. They have been normalized against protein
concentration by using the Bradford Protein Assay (Bio-Rad).

Urea production in H9 NHSM-Hep cells was lower than the H9-Hep cells. In H9-
Hep cells, there is a slight increase in urea production when the cells are exposed to
17
increased concentrations of ammonia. On the other hand, there is no change in urea
production when H9 NHSM-Hep cells are exposed to increased concentrations of
ammonia. This could mean that the differentiated H9 cells are more developed than the
differentiated H9 NHSM cells, making them slightly more mature. Hepatocytes produced
more urea when they were exposed to 5mM of ammonia. Like the results in the
previous assays, the differences were not statistically significant between H9-Hep cells
and H9 NHSM-Hep cells.
4.5 Albumin ELISA
H9-Hep
H9 NHSM-Hep
Hepatocytes
[Albumin] (ng/ml)

Figure 8. Albumin secretion by the differentiated cells at Day 10 and Day 20. They have
been normalized against protein concentration by using the Bradford Protein Assay
(Bio-Rad).
18
Between H9-Hep cells and H9 NHSM-Hep cells, it seems the former produced
more albumin. Once again, the difference is not significant between the two cell types.
However, the concentration of albumin secreted by both cell types is significantly higher
than hepatocytes. This could be due to the in vitro culture of hepatocytes. When
hepatocytes are cryopreserved and then thawed for use, the optimal recovery of their
functions can vary [18]. However, even though the difference in albumin concentration
between the differentiated cells and hepatocytes is high, the data still show that there
was an increase in albumin secretion throughout the differentiation process.
4.6 PAS Staining

Figure 9. Glycogen storage by the differentiated cells. Images were taken at 5x.

The hepatic function of glycogen storage can be seen occurring in some of the
differentiated cells. Some of the stained cells show hepatic progenitor cell morphology
where they are clustered and polygonal in shape. However, these cells cannot be
determined as true hepatocytes unless further assays are performed. To do this, we can
try staining the cells for albumin and HNF4a markers.

19
Chapter V
Discussion and Conclusion
Discussion
The study of human naïve stem cells is still relatively new and many groups are
currently trying to find methods of inducing human embryonic stem cells into their naïve
state. In Hanna’s lab, human naïve stem cells were obtained by culturing cells under
conditions that contained many small molecules that affects many different signaling
pathways. Although the mechanisms of these molecules and their cascading pathway
effects on cell differentiation are still not fully understood, many believe that achieving
human naïve stem cells can lead to greater therapeutic potentials similar to the goals of
differentiating human embryonic stem cells.
Therefore, this study was designed to compare the yield of hepatocyte-like cells
between human naïve stem cells and hESCs. The potential in the hepatic differentiation
of these cells was determined by observing their gene expressions of hepatocyte-
specific genes as well as the ability to function like hepatocytes. Due having more
mESC-like characteristics, it was hypothesized that human naïve stem cells can more
efficiently differentiate to hepatocyte-like cells that human embryonic stem cells.
We differentiated H9 NHSM cells and H9 cells using the protocol mentioned
previously. However, results showed that there were no significant differences in
differentiating human naïve stem cells and human embryonic stem cells. Although the
differentiated human naïve stem cells had higher relative gene expressions in albumin
and factor X compared to differentiated human embryonic stem cells while they had
20
lower gene expressions in HNF4a, tendencies of human embryonic stem cells
displaying more hepatocyte-like characteristics can be observed. This could be due to
the protocol used.
The protocol used to differentiate both cell types may be in favor of human
embryonic stem cells since these protocols were developed using only human
embryonic stem cells and not human naïve stem cells [3]. In addition, naïve stem cells
are considered to be at an earlier stage of the differentiation process compared to the
stem cells in the primed state [6]. Thus, there may be extra stages naïve stem cells
need to go through in order to fully differentiate to mature cells. This could explain why
the morphological change in the differentiated cells at day 20 shows more immature
hepatoctyte-like shapes in H9 NHSM-Hep cells while the H9-Hep cells show more
mature hepatocyte-like shapes. Therefore, the differentiation protocol for human naïve
stem cells needs to be further explored.
The PAS staining results previously shown indicates that some cells may have
developed the hepatic function to store glycogen, yet their morphology look more like
progenitor hepatic cells. Once again, this could be due to the protocol used for the
differentiation process. Since a kit was used to differentiate the stem cells to the
definitive endoderm stage, the contents are not disclosed. That could have contributed
to the formation of cells into different lineages, For example, extraembryonic lineage
such as trophoblasts. Yet, the kit was used for differentiation to the definitive endoderm
because it was observed that this method allowed for more cell survival compared to
using another method that involved the use of activin A and BMP4 on hESCs.
21
Furthermore, recent studies have debated that the use of BMP 4 can induce
differentiation of human embryonic stem cells to trophoblasts. If this is the case, the use
of BMP4 in the hepatic differentiation protocol could have had an influence on the cell
lineages [15]
Comparing both H9- and H9 NHSM-Hep cells to primary hepatocytes, it can be
seen that the differentiated cells hepatocyte-like characteristics have yet to reach the full
mature hepatocyte capabilities. The time allowed for differentiation in the protocol can
play a role in the cells maturation to hepatocyte-like cells. Although in normal human
fetal liver development, the liver primordial appears around 21 days of gestation where
mature hepatocytes have not yet formed in the embryo [14]. Yet, most standard
protocols for hepatic differentiation in hESCs induce differentiation for around 20 days
[12]. These 20 days may not be enough time to let the cells fully differentiate into
mature hepatocytes.
Essentially, if a protocol for hepatic differentiation can be catered to human naïve
stem cells, or improved for human embryonic stem cells, the resources for liver models
can be expanded and allow for easier access.
Conclusion
Due to their mESCs-like characteristics, we hypothesized that human naïve stem
cells would efficiently differentiate to hepatocyte-like cells compared to hESCs.
However, even though the differences weren’t significant, this study demonstrated that
hESCs that underwent hepatic differentiation had a higher tendency to show more
hepatic characteristics compared to human naïve stem cells that have undergone the
22
same differentiation process. Again, this could be due to the use of hepatic
differentiation protocols that have been established for hESCs as well as the shortened
differentiation time compared to the in vivo time. Further studies would need to be done
to explore the potential of human naïve stem cells and their hepatic differentiation
efficiencies. By doing so, the resource for liver disease modeling and therapeutic efforts
can be expanded and easily accessed.

23
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Abstract (if available)

Abstract With the potential of using stem cells to provide a large source of hepatocytes for liver modeling or cell therapy, human naïve stem cells are becoming the topic of many studies. Past studies have divided stem cells to two states: naïve and primed. Since the stem cells in mice are pluripotent and considered less advanced in the differentiation process, they are considered in the naïve state. On the other hand, human embryonic stem cells show characteristics of being in the primed state. Thus, studies have reprogrammed human embryonic stem cells into a naïve state to reflect the mouse embryonic stem cell-like characteristics. In our study, we induced hepatic differentiation in both human embryonic stem cells and human naïve stem cells to compare their yield of hepatocyte-like cell. Because of the mouse embryonic stem cell-like characteristics, we hypothesized that the human naïve stem cells would efficiently differentiate to hepatocyte-like cells compared to human embryonic stem cells. A modified standard protocol was used to differentiate the cells and the resulting cells were checked for hepatic characteristics. The results of this study showed that there were no significant differences in the yield of hepatocyte-like cells. However, the differentiated human embryonic stem cells had a tendency to be slightly more mature than the differentiated human naïve stem cells. Further studies are needed to explore the cause for this and to optimize hepatic differentiation in both cell types.

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Asset Metadata

Creator Nguyen, Samantha K. (author)

Core Title Hepatic differentiation in human naïve stem cells compared to human embryonic stem cells

School Keck School of Medicine

Degree Master of Science

Degree Program Molecular Microbiology and Immunology

Publication Date 07/24/2015

Defense Date 06/16/2015

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag embryonic stem cells,ground state,hepatic differentiation,hepatocyte,human naïve stem cells,mouse embryonic stem cells,naïve human state media,naïve state,OAI-PMH Harvest,primed state,stem cells

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Schonthal, Axel (committee chair), DePaolo, R. William (committee member), Machida, Keigo (committee member), Miki, Toshio (committee member)

Creator Email samantkn@usc.edu,sammyknguyen@sbcglobal.net

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-605303

Unique identifier UC11300289

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Legacy Identifier etd-NguyenSama-3692.pdf

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Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

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