Induction of Highly Functional Hepatocytes from Human Umbilical Cord Mesenchymal Stem Cells by HNF4α Transduction
et al. (2014) Induction of Highly Functional Hepatocytes from Human Umbilical Cord Mesenchymal Stem Cells by
HNF4a Transduction. PLoS ONE 9(8): e104133. doi:10.1371/journal.pone.0104133
Induction of Highly Functional Hepatocytes from Human Umbilical Cord Mesenchymal Stem Cells by HNF4a Transduction
Hualian Hang 0
Yabin Yu 0
Ning Wu 0
Qingfeng Huang 0
Qiang Xia 0
Jianmin Bian 0
Graca Almeida-Porada, Wake Forest Institute for Regenerative Medicine, United States of America
0 1 Department of Liver Surgery, Ren Ji Hospital, School of Medicine, Jiao Tong University , Shanghai , China , 2 Department of General Surgery, Nanjing Hospital Affiliated to NanJing Medical University , Nanjing , China
Aim: To investigate the differentiation potential of human umbilical mesenchymal stem cells (HuMSCs) and the key factors that facilitate hepatic differentiation. Methods: HuMSCs were induced to become hepatocyte-like cells according to a previously published protocol. The differentiation status of the hepatocyte-like cells was examined by observing the morphological changes under an inverted microscope and by immunofluorescence analysis. Hepatocyte nuclear factor 4 alpha (HNF4a) overexpression was achieved by plasmid transfection of the hepatocyte-like cells. The expression of proteins and genes of interest was then examined by Western blotting and reverse transcription-polymerase chain reaction (RT-PCR) or real-time RT-PCR methods. Results: Our results demonstrated that HuMSCs can easily be induced into hepatocyte-like cells using a published differentiation protocol. The overexpression of HNF4a in the induced HuMSCs significantly enhanced the expression levels of hepatic-specific proteins and genes. HNF4a overexpression may be associated with liver-enriched transcription factor networks and the Wnt/b-Catenin pathway. Conclusion: The overexpression of HNF4a improves the hepatic differentiation of HuMSCs and is a simple way to improve cellular sources for clinical applications.
Funding: Project supported by the National Natural Science Foundation of China (No 81100306)and the Science and Technology Commission Medical
Foundation of Shanghai (No. 134119a9501). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the
Competing Interests: The authors have declared that no competing interests exist.
. These authors contributed equally to this work.
Most liver diseases lead to hepatocyte dysfunction with possible
eventual organ failure. Bioartificial livers and hepatocyte
transplantation have been regarded as useful bridges for patients
waiting for whole-organ transplantation [1,2] by providing
metabolic support during liver failure. However, organ shortage
remains a major limiting step of this procedure. More recent
developments in stem cell technology have highlighted a new
source of liver cells for use in regenerative medicine. In particular,
mesenchymal stem cells (MSCs), due to their multipotency and
low immunogenicity, have been suggested as an ideal population
of stem cells for the treatment of liver injury. Human umbilical
cord mesenchymal stem cells (HuMSCs) isolated from umbilical
cord Whartons jelly are more primitive MSCs than those isolated
from other tissue sources and do not express the major
histocompatibility complex (MHC) class II (HLA-DR) antigens
[3,4]. However, the hepatic differentiation status of
hepatocytelike cells derived from stem cells is not sufficient for clinical use
because of the relatively low expression levels of functional
proteins and the lack of full induction of metabolic activity .
Therefore, it is important to define a method to promote hepatic
Hepatocyte differentiation is associated with changes in gene
expression that are primarily controlled at the level of
transcription. Studies of the transcriptional gene regulatory elements
expressed in hepatocytes have identified a number of liver
transcription factors, including hepatocyte nuclear factor
(HNF)1, -3, -4, and -6 and the CCAAT/enhancer-binding protein
(CEBP) family, that are capable of modulating hepatocyte gene
expression in hepatoma cells [6,7]. Hepatocyte nuclear factor 4
alpha (HNF4a), an important transcription factor of the nuclear
hormone receptor family, is essential for normal liver architecture,
the morphological and functional differentiation of hepatocytes,
and the generation of the hepatic epithelium [8,9]. Several studies
have demonstrated that HNF4a may act as a master gene in a
transcription factor cascade that could drive hepatic differentiation
[10,11]. The overexpression of HNF4a could improve the
hepatocyte functions of hepatocyte-like cells derived from human
embryonic stem cells, induced pluripotent stem cells and bone
marrow mesenchymal stem cells [12,13]. In this study, we
demonstrate that HuMSCs can be differentiated into
hepatocyte-like cells according to the protocol established by Lee et al.
. The overexpression of HNF4a can significantly improve the
differentiation status of hepatocyte-like cells through the activation
of several target genes. These more differentiated hepatocyte-like
cells can provide a better cell source for future clinical applications.
Materials and Methods
Isolation and culture of HuMSCs
With the informed consent of the tissue donor, and following the
ethical and institutional guidelines, fresh human umbilical cords
were obtained from male or female fetuses after birth, and 20
cords were collected in our experiment. The study was approved
by the Institution Review Board and Human Ethics Committee of
Affiliated Nanjing Hospital of Nanjing Medical University,
Jiangsu, China. Written consent for the use of the samples for
research purposes was obtained from all of the included patients.
The samples were then maintained in phosphate-buffered saline
(PBS; Invitrogen, Carlsbad, CA, USA) containing 100 U/mL
penicillin(Sigma-Aldrich, St Louis, MO, USA) and 100 mg/mL
streptomycin(Sigma-Aldrich) at 4uC. Following disinfection in
75% ethanol for 1 min, the umbilical cord vessels were removed in
PBS. HuMSCs were prepared as previously described . The
mesenchymal tissue was diced into cubes of approximately 1 cm3.
Following the removal of the supernatant fraction, the precipitate
was washed with DMEM (Invitrogen) and centrifuged at 2506g
for 5 min. The mesenchymal tissue was treated with collagenase II
(Invitrogen) at 37uC for 1 h and further digested with 0.25%
trypsin (Invitrogen) at 37uC for 30 min. Fetal bovine serum (FBS;
Invitrogen) was added to the mesenchymal tissue to neutralize the
excess trypsin. The dissociated mesenchymal cells were further
dispersed by treatment with 10% FBS-DMEM and counted. The
mesenchymal cells were then used directly for cultures, and the
medium was changed twice a week.
Flow cytometry analysis
Antibodies against the human antigens CD13, CD105, CD90,
CD34, CD45, HLA-DR and HLA-DQ were purchased from BD
Sciences (Shanghai, CHINA). A total of 16106 cells were
resuspended in 200 mL of PBS and incubated with FITC- or
PE-conjugated antibodies for 30 min at room temperature. The
fluorescence intensity of the cells was evaluated by flow cytometry
using a flow cytometer (FACScan; BD Sciences), and the data
were analyzed using CELLQUEST Pro software (BD Sciences).
To induce adipogenic differentiation, the cells were treated with
adipogenic medium for three weeks with medium changes twice
weekly. Briefly, after the cells reached 70% confluence, the
medium was changed to adipogenic differentiation medium
consisting of L-DMEM supplemented with 10% FBS, 2 mM
IBMX (Sigma-Aldrich) and 5 mg/mL insulin solution
(SigmaAldrich). The generation of lipid vacuoles was visualized by
staining with Oil Red O (Sigma-Aldrich).
The pancreatic induction of HuMSCs was performed according
to the procedure developed by Wang et al . The cells were
cultured for seven days in DMEM/F12 (Invitrogen) medium
containing 10% FBS, 4 nM activin-A (Sigma-Aldrich), 10 mM
nicotinamide (Sigma-Aldrich) and 25 ng/mL epidermal growth
factor (EGF, PeproTech, Rocky Hill, NJ, USA). The culture
medium was changed to DMEM/F12 for seven days. Finally,
10 mM nicotinamide, insulin/transferring/selenium (ITS,
Invitrogen), and 10 ng/mL basic fibroblastic growth factor (bFGF,
PeproTech) were added, and the incubation was continued for 17
days. After induction, the cells were stained with dithizone.
To induce Chondrogenic differentiation, the 4th passage cells
were treated with Chondrogenic medium for 21 days (A1007101
STEMPRO CHONDRO DIFF KIT, GIBCO). Medium changes
were performed twice weekly, and chondrogenesis was assessed by
immunohistochemical staining for type II collagen.
Hepatic differentiation of HuMSCs
According to a previously described protocol , the stem cells
were deprived for two days in IMDM supplemented with 20 ng/mL
EGF, 10 ng/mL bFGF and 0.61 g/mL nicotinamide for seven days.
The cells were then treated with step-2 maturation medium consisting
of IMDM supplemented with 20 ng/mL oncostatin M (OSM,
PeproTech), 1 mmol/L dexamethasone (DEX, Sigma-Aldrich), and
50 mg/mL ITS. The medium was changed twice per week.
The cells were fixed in PBS containing 4% paraformaldehyde
(NJ-reagent, NanJing, CHINA) for 30 min and permeabilized
with phosphate-buffered saline containing 0.1% Triton X-100
(NJ-reagent) for 20 min. The samples were incubated with
antiHNF4a antibody (1:200; Santa Cruz Biotechnology, Santa Cruz,
CA, USA), anti-human serum AFP antibody (1:200; Santa Cruz
Biotechnology), and anti-human serum ALB antibody
(1:200;Santa Cruz Biotechnology), and then with the secondary
antibody conjugated to fluorescent phycobiliproteins, namely
DyLight 594- and Alexa 488-conjugated goat anti-mouse
immunoglobulin G (1:1000; Beyotime, Shanghai, China). DAPI
(Beyotime) was used for nuclear counterstaining.
Intracellular glycogen was analyzed by Periodic AcidSchiff (PAS)
staining. Culture dishes containing the cells were fixed in 4%
paraformaldehyde and permeabilized with 0.1% Triton X-100 for
10 min. The samples were then oxidized in 1% periodic acid for
10 min, rinsed 3 times in deionized water (dH2O), treated with Schiff
s reagent for 20 min at room temperature and rinsed in dH2O for 5
to 10 min. The nuclei were stained with Mayers hematoxylin for
1 min, rinsed in dH2O and assessed under a light microscope.
The concentration of human Albumin protein on was assayed
by a quantitative enzyme-linked immunosorbent assay kit (ELISA)
(R&D Systems, USA).
Western blot analysis
The western blot analysis was performed as described previously
. The total cellular protein was extracted using cell lysis buffer
(KeyGEN, Nanjing, China). The proteins were separated by
electrophoresis (Bio-Rad, CA, USA) and transferred to
membranes (NJ-reagent). The membranes were blocked in blocking
solution (NJ-reagent) and incubated with mouse monoclonal
Annealing temperature (6C)
antibodies against alpha-fetoprotein (AFP), albumin (ALB) and
cytochrome P450 3A4 (CYP3A4, 1:200; Santa Cruz
Biotechnology) for 1 h at room temperature. After washing, the membranes
were incubated for 2 h with horseradish peroxidase (HRP)-linked
goat anti-mouse IgG (1:1000; Biosynthesis, Beijing, China). The
membranes were rinsed for 10 s in substrate buffer (NJ-reagent) to
remove any residual detergent. A mouse monoclonal antibody
against b-actin (1:5000; KeyGEN) was used as a housekeeping
Reverse transcription-polymerase chain reaction and
real-time reverse transcription-polymerase chain reaction
The total RNA from the cells was isolated using the TRIzol
reagent (Invitrogen) according to the manufacturers instructions.
The cDNA templates were obtained using oligo(dT) primers and
Construction of the PCDNA3.1/HNF4a recombinant
We generated the HNF4a cDNA using specific primers
59-AGGATCCATGCGACTCTCCAAAACCCTCG-39; reverse primer:
59-AGAATTCCCTAGATAACTTCCTGCTGCTTGGTG-39). The sequence was then inserted into
PCDNA3.1 (Invitrogen), resulting in the generation of
PCDNA3.1/HNF4a. The element was confirmed by digesting
with EcoR I (TaKaRa, Shiga, Japan) and BamH I (TaKaRa). The
two-week-induced MSCs were transfected with the recombinant
plasmid using Lipofectamine 2000 (Invitrogen). The target gene
overexpression was confirmed by RT-PCR and
The statistical significance of the induction effect was
determined by t-tests. Differences were considered significant if the P
value was less than 0.05.
Isolation and culture of HuMSCs
The isolated cord cells manifested heterogeneity during the first
five days. Forty-eight hours after plating, the cells were adherent,
elongated and spindle-shaped, and the individual colonies formed
displayed a fibroblast-like morphology (Fig. 1A1). The primary
culture cells reached confluence approximately two weeks later,
and the cells were then passaged at a ratio of 1:3. The cultured
HuMSCs were positive for CD13, CD90, CD105 and CD59 but
negative for hematopoietic makers, including CD34, CD45 and
HLA-DR (Fig. 1B). The HuMSCs were investigated to determine
their potential for mesodermal and endodermal differentiation. Oil
Red O staining revealed that the HuMSCs contained positively
stained lipid droplets in the cytoplasm after adipogenic
differentiation (Fig. 1A3). Positive dithizone staining indicated that the
HuMSCs could differentiate into pancreatic islet-like cells
(Fig. 1A5). Immunohistochemical staining for type II collagen
revealed that the HuMSCs could differentiate into cartilage cells
after chondrogenic differentiation (Fig. 1A7). Undifferentiated
HuMSCs cultured in the growth medium did not show staining
for Oil Red O, dithizone or type II collagen. (Fig. 1A2, A4, A6).
Differentiation status of hepatocyte-like cells induced
Under hepatic induction conditions, the fibroblastic
morphology of HuMSCs gradually changed toward the polygonal shape of
hepatocytes with the appearance of abundant granules in the
cytoplasm (Fig. 2A).In addition to the morphological differences,
we detected hepatocyte-specific marker expression by
immunofluorescence. After the HuMSCs were incubated in hepatocyte
differentiation medium for 21 days, they became positive for ALB
and AFP. The cells cultured in growth medium, which were used
as negative controls, were not positively stained for ALB and
exhibited low AFP expression (Fig. 2B).
Transduction of HuMSC-derived hepatoblasts with
HNF4a efficiently promotes hepatic maturation
We aimed to determine whether hepatic maturation is
promoted by PCDNA3.1/HNF4a transduction. The human
HNF4a gene was introduced into nine-day-induced HuMSCs by
plasmid transfection. The overexpression of HNF4a was examined
by RT-PCR (Fig. 2D) and immunofluorescence (Fig. 2C)
compared to mock control cells infected with PCDNA3.1. The
transduced cells were cultured until day 21 of differentiation
according to the schematic protocol described in Figure 3A. The
control group was transfected with PCDNA3.1 alone and
maintained in differentiation medium (DM). We examined the
hepatic gene expression on day 21 of differentiation by real-time
RT-PCR. The gene expression analysis of TAT, ALB, G-6P,
CYP3A4 and a1AT in the transfected HNF4a group revealed
higher levels of these genes compared to those found in the cells
maintained in hepatic differentiation medium, whereas AFP
exhibited lower expression (Fig. 3C). We also examined the
protein levels of ALB, AFP and CYP3A4 by western blotting
(Fig. 3B), and the results were consistent with the real-time
RTPCR findings. Upon treatment with PCDNA3.1/HNF4a
transduction, glycogen uptake was detected after 21 days by Periodic
Acid-Schiff (PAS) staining (Fig. 4A). Intracellular glycogen in the
transfected HNF4a group (Fig. 4A3) was higher levels compared
to control group (Fig. 4A2). Both groups were negative for MHC
(HLA-DR and HLA-DQ) after 21days (Fig. 4B). Albumin was
secreted in the culture media and was analyzed on days 0, 3, 6, 9,
12, 15, 18 and 21 of differentiation. We examined the transfected
HNF4a group produced significantly higher levels of albumin
compared to control group from about 12 days of differentiation
(Fig. 4C). From the results of FACS, about 98% of the cells in
transfected HNF4a group expressing ALB, whereas the control
group was 82% (Fig. 4D).
HNF4a promotes hepatic differentiation by regulating
Several studies have demonstrated that hepatic gene expression is
regulated by the combinational action of liver-enriched factors. In this
study, we observed weak expression of HNF4a, HNF3b, HNF6 and
CEBP/a in the cells cultured in differentiation medium. HNF4a is
crucial for the expression of hepatic genes, including liver-enriched
transcription factors . Therefore, we investigated whether the
transcriptional efficiency of liver-enriched factors was regulated by
HNF4a overexpression in the induced HuMSCs. The expression
levels of additional liver-enriched transcription factors in the
HNF4atransfected cells were examined by RT-PCR analysis. HNF6 and
CEBP/a exhibited robust expression three days after transfection.
This increase continued for 12 days after transfection, and HNF3b
began to be expressed at high levels (Fig. 5A).Besides, we also analysis
of Wnt/b-catenin in hepatocyte-like cells after HNF4a transfection.
Because the Wnt/b-catenin pathway plays a fundamental role in the
control of adult stem cell differentiation, we analyzed the expression
of several genes regulated by this pathway, such as b-catenin,
glycogen synthase kinase 3 beta (GSK3b), dickkopf 4 (DKK4) and
frizzled 2 (FZD2). The results indicated that this pathway was
suppressed upon induction in hepatic differentiation medium, and
this effect was more pronounced after HNF4a transfection (Fig. 5B).
Many reports have demonstrated the pluripotency and low
immunogenicity of HuMSCs; thus, these cells are expected to be a
source of specific cell types for transplantation. The phenotypic
profile of the HuMSCs isolated in our study was consistent with
that found in previous studies and the documented expression of
the consensus MSC marker set. Indeed, the HuMSCs were
positive for CD90, CD105, CD13 and CD59 and negative for
CD45, CD34 and HLA-DR (Fig. 1B). The HuMSCs were
examined for their ability to undergo adipogenic and pancreatic
differentiation to illustrate their multipotent differentiation
potential for not only mesodermal but also endodermal differentiation.
Our results also demonstrated that the HuMSCs were well
differentiated (Fig. 1A).
The hepatic differentiation of HuMSCs has been reported by
the addition of various growth factors and cytokines. In this study,
we induced the hepatic differentiation of HuMSCs according to
the protocol described by Lee et al. . The hepatic gene
expression pattern in the induced MSCs appears to be correlated
with the developmental process of the liver in vivo. We found that,
upon exposure to the differentiation medium for two weeks, the
cells began to form clusters (Fig. 2A). Then, the HuMSCs
gradually progressed toward the polygonal morphology of mature
hepatocytes and expressed liver-specific protein markers, such as
ALB and AFP (Fig. 2B). However, the differentiation efficiency
remains insufficient for clinical application. Therefore, we
attempted to achieve transdifferentiation with high efficiency by
overexpressing HNF4a, which plays an important part in hepatic
differentiation. HNF4a is initially expressed in the developing
hepatic diverticulum on E8.75 [18,19], and its expression is
elevated as the liver develops. A previous loss-of-function study
showed that HNF4a plays a critical role in liver development; the
conditional deletion of HNF4a in fetal hepatocytes results in the
faint expression of many mature enzymes and the impairment of
normal liver morphology . Our study first focused on the
function of HNF4a in the hepatic differentiation of HuMSCs.
According to Takayama et al. , we chose the optimal time
for HNF4a transfection as the time at which the cells were induced
into hepatoblasts. Because endogenous HNF4a is initially
expressed in the hepatoblasts [18,19], our system might adequately
reflect early embryogenesis (Fig. 3A). We found that the
expression levels of the functional hepatic genes TAT, ALB, CYP3A4,
G-6P and a1-antitrypsin were upregulated by HNF4a transfection
compared to cells differentiated only in hepatic differentiation
medium, whereas AFP exhibited lower expression, indicating a
higher degree of hepatocyte maturation (Fig. 3C). The results of
the western blot analyses of the ALB, CYP3A4 and AFP proteins
were consistent with the real-time RT-PCR findings.
It is well known that several liver-enriched transcription factors
can coordinately regulate the expression of hepatic genes involved
in liver-specific functions [20,21]. Among them, HNF4a may act
as a master gene in the transcriptional cascade that regulates the
constitutive expression of target genes. Therefore, we investigated
Figure 4. Hallmark function assays of mature hepatocytes. (A) Periodic Acid-Schiff staining assay in hepatogenic differentiated cells.
A1:HuMSCs; A2: HuMSCs were cultured in hepatocyte differentiation medium for 21 days; A3: HuMSCs were transfected with HNF4a plasmid and
cultured until day 21. (B) Expression of MHC (HLA-DR and HLA-DQ) assay by flow cytometry analysis. B1,B4:HuMSCs;B2,B5: HuMSCs were cultured in
hepatocyte differentiation medium for 21 days transfected with PCDNA3.1 alone;B3,B6: HuMSCs were transfected with HNF4a plasmid and cultured
until day 21. (C, D) Albumin levels in hepatogenic transfected with HNF4a differentiated cells in comparison to cells cultured in differentiation
medium for 21 days transfected with PCDNA3.1 alone by enzyme linked immunosorbent assay (P,0.05) and Flow cytometry analysis (D1: control
group D2:HNF4a group).
the transcriptional efficiency of liver-enriched factors regulated by
HNF4a overexpression in induced HuMSCs. Interestingly, we
found that the HuMSCs weakly expressed HNF4a but did not
express other liver-enriched factors.
Previous studies have demonstrated that HNF4a is expressed
not only in the liver but also in the kidney, heart, spleen and
intestine, although at different levels [22,23]. The overexpression
of HNF4a before hepatic specification may promote bidirectional
differentiation and a heterogeneous population. Therefore, we
chose the appropriate time at which HNF4a could participate in
the liver-enriched factor network. After the cells were induced in
hepatic differentiation medium for nine days, the liver-enriched
factors gradually began to be expressed (Fig. 4A). However, the
factors were still weakly expressed until 21 days after induction.
The overexpression of HNF4a upregulated the expression of
HNF6, CEBP/a and HNF3b after transfection, as determined by
the RT-PCR analysis results. Therefore, we concluded that
HNF4a might promote hepatic differentiation by regulating
liver-enriched factors. Wnt/b-catenin is activated as mesenchymal
stem cells differentiate into osteoblasts  and is inactivated
during differentiation into adipocytes . Previous reports have
shown that the down-regulation of Wnt/b-catenin signaling plays
a role in the hepatic differentiation of MSCs . We also
focused on Wnt/b-catenin signals as one of the important
mechanisms for hepatic differentiation induced by HNF4a
overexpression. Our results showed that the overexpression of
HNF4a suppressed this pathway to a greater extent than observed
in cells induced in hepatic differentiation medium alone. Thus, the
improved hepatic differentiation of HuMSCs caused by HNF4a
may be associated with the inactivation of Wnt/b-catenin signals.
In summary, our data demonstrate that HuMSCs can be easily
induced into hepatocyte-like cells under hepatic differentiation
conditions. Furthermore, HNF4a is a key factor in determining
the differentiation status of the hepatocyte-like cells derived from
HuMSCs. The overexpression of HNF4a can activate various
hepatic-specific genes and enhance the differentiation status of
differentiated HuMSCs. This research provides an experimental
basis for further research in stem cell transplantation, and stem cell
transplantation will provide a broad scope in future clinical
applications for liver regeneration after hepatectomy and living
donor liver transplantation (LDLT). This investigation may also
provide the means to generate reliable cell sources for bioartificial
liver support devices and hepatocyte transplantation in the future.
However, several limitations, including genomic integration into
target cells and the cytotoxicity of target cells, have impeded the
clinical utility of these methods. Therefore, solutions should be
considered in future studies.
Conceived and designed the experiments: JB QX. Performed the
experiments: HH YY. Analyzed the data: NW. Contributed reagents/
materials/analysis tools: QH. Wrote the paper: HH.
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