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Identification of Cytochrmoe oxidase p450 in Heptocytes generated from
in vitro differentiation of mouse mesenchymal stem cells
Zahraa K. Zedan1 Shalal M. Hussain2 Shahlaa M. Salih 3
1Al-Nahrain University, College of Science, Deparetemnt of Biotechnology, Baghdad, Iraq
zahraa.alfahad@yahoo.com
2Al- mustansyria University , Faculty of Pharmecy, Baghdad, Iraq
dr_shalal@yahoo.com
3Al-Nahrain University, College of Sience, Department of Biotechnology, Baghdad, Iraq
shahlaam2006@yahoo.com
ABSTRACT
This study was designed to identify the presence of cytochrome oxidase p450 (CYP3A4), in the hepatic cell lines which
was generated from the in vitro differentiation of mouse bone marrow mesenchymal stem cells. Bone marrow meenchymal
stem cells were first isolated by collecting both the femur and tibia of the mouse thigh bone, then exess tissue were
removed and the cells were flushed from the bones and cultured under highly aseptic conditions. The isolated cells were
characterized as a mesenchymal stem cells via using immunocytochemistry analysis. The characterized hepatocytes were
subjected to a differentiation protocol in which the differentiation medium was contained a spesfic growth factor including
(Hepatocytes growth factor (HGF), Fibroblast growth factor4 (FGF4), Oncostatin M and Dexamethasone, All theses
growth factor was used for directing the BM-MSCs twards the hepatic line of cells. After three weeks of the differentiation
protocol the diffrentation cells were examined for the presence of Cytochrome oxidase p450 (CYP3A4), which consider as
one of the specific markers of hepatocytes. Identification eas done via two different methods first, via using the
immunocytochemical analysis for the antihuman mouse CYP3A4 antibody and second by ELIZA test. The results showed
a posotive reactivity of the differentiated cells towards the mouse antihuman CYP3A4 antibody as well as the eliza results
showed a significant increase in the enzyme level of the cell lysate during the weeks of the differentiation experiment.
Keywords: Cytochrome oxidase p450; mesenchymal stem cells; hepatocytes;
Academic Discipline And Sub-Disciplines
Stem cell technology
SUBJECT CLASSIFICATION
Regenerative medicine Classification
TYPE (METHOD/APPROACH)
Immunocytochemistry, Elisa analysis
Council for Innovative Research
Peer Review Research Publishing System
Journal of Advances in Biology
Vol. 7, No. 3
www.cirjab.com
editorsjab@gmail.com , editor@cirjab.com
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INTRODUCTION
Cytochrome P450 3A4 (abbreviated CYP3A4) is an important enzyme in the body, mainly found in the liver and in the
intestine. Its purpose is to oxidize small foreign organic molecules (xenobiotics), such as toxins or drugs, so that they can
be removed from the body [1]. The potential ability of CYP3A4 to oxidize molecules with widely diverse in size and
chemical structure is due to its large and malleable active site of the detoxitative molecule [2]. Another intriguing feature of
CYP3A4 is the ability to accommodate more than one molecule in the substrate-binding pocket, where one molecule
serves as a substrate while another acts as a modulator of substrate metabolism. Among the substrates that exhibit
binding cooperativity with CYP3A4 are testosterone, progesterone, diazepam, α-naphthoflavone, and others [3].While
many drugs are deactivated by CYP3A4, there are also some drugs which are activated by the enzyme. Some
substances, such as grapefruit juice and some drugs, interfere with the action of CYP3A4. These substances will therefore
either amplify or weaken the action of those drugs that are modified by CYP3A4 [4].The enzyme CYP3A4 is a member of
the cytochrome P450 family of oxidizing enzymes. Several other members of this family are also involved in drug
metabolism, but CYP3A4 is the most common and the most versatile one. Like all members of this family, it is a
hemoprotein, i.e. a protein containing a heme group with an iron atom. In humans, the CYP3A4 protein is encoded by the
CYP3A4 gene, this gene is part of a cluster of cytochrome P450 genes on chromosome 7 [5].
MSCs are a type of multipotent adult stem cells that can be readily obtained from BM aspirates and expanded into large
quantities in vitro. Furthermore, MSCs possess potent immunosuppressive activities. Therefore MSCs could be used in
patients who are in need of immune modulation, as well as tissue repair, such as organ transplant recipients and patients
with severe autoimmune diseases [6]. MSCs considered as the ideal cells in the therapeutic approaches. The
differentiation of MSCs is regulated by various signals from microenvironment, which is consisting of many biological
molecules and biomechanical forces. These factors play a vital role in determining the efficacy of MSCs biology [7].
A major area of research in transplantation medicine is the potential application of stem cells in liver regeneration. This
would require well-defined and efficient protocols for directing the differentiation of stem cells into the hepatic lineage,
followed by their selective purification and proliferation in vitro.
MATERIALS AND METHODS
Experimental animals
Swiss albino mice of four to eight weeks old were obtained from the Laboratory Animal Unit of the Iraqi center for cancer
researches and medical genetics Baghdad, Iraq were used in this research. The animal housing were performen under a
clean conditions of healthy diet and acycle of 12 hrs day and light.
Isolation of bone marrow MSCs
The mouse bone marrow MSCs (mBM-MSCs) were isolated by collecting the thigh bones. The bone marrow was extruded
by clipping of the epiphyseal ends of both femur and tibia bones and flushing with Minimum essential medium (MEM)
(Sigma, St. Louis, MO), supplemented with 10% fetal bovine serum (US Biological, USA), 1% penicillin/streptomycin
(Medium A). After 24 hours, non-adherent cells and debris were removed, and the adherent cells were cultured
continuously. At near confluence, the cells were replated at 5 × 104 cells/ml [8].
Characterization of mouse MSCs
Isolated MSCs were identified from the other bone marrow cells haematopoietic stem cells using immunocytochemical
analysis, which based on the cell nature of expressing specific cell markers on their surface. These techniques are based
on the immunoreactivity of antibodies and the chemical properties of enzymes or enzyme complexes which react with
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colorless substrate- chromogens to produce a colored end product. Initial immuno-enzymatic stains utilized the direct
method, which conjugates enzymes directly to an antibody with known antigenic specificity (primary antibody). It allowed
for the visualization of tissue antigens using a standard light microscope [13].
Induction of hepatogenic differention
In hepatogenic differentiation the 3rd passage of (2 ×104) of mBM-MSCs were used. The differentiation strategy involved
two main stages in first stage which lasted for a week the cells were cultured in a DMEM medium supplemented with 10%
FBS, 20 ng/ml (Fetal bovine serum) HGF, 20 ng/ml FGF4 (Fibroblast growth factor) and 10-7 M/L dexamethasone. At the
second stage which lasted for two weeks the same media where used as the first stage with the addition of 10 ng/ml of
oncostatin M (Sigma, USA). As a negative control, mBM-MSCs were cultured in medium without differentiation stimuli
along with the differentiation experiments in the same conditions. Cells were cultured in a humidified atmosphere of 5%
CO2 and 95% air at 37℃. Cultures were maintained by medium exchange every 3 d. The cell morphology was observed
under inverted microscope [6].
Identification of CYP3A4 in differentiated cells via immunocytochemical anlyais
After three weeks of differentiation, the cultured cells were washed twice with PBS and fixed with 4% paraformaldehyde
for 30-45 min at room temperature and permeabilized with 0.4% Triton X-100 (Sigma-Aldrich, St. Louis, USA) for 10 min.
After blocking with bovine serum albumin, the washed cells were incubated overnight at 4 °C with primary antibody which
is a mouse anti-human CYp3A4 (1:1000) [4]. The cells were then incubated with DAPI stain (4’,6-diamidino-2-
phenylindole; 1:1000) for nuclear staining, between each incubation, the samples were washed with PBS-0.05% Tween.
Then the counter stain heamatotoxilin stain is added. The ratio of immunopositive cells to the total number of cell nuclei
labeled with DAPI was recorded [7].
Identification of CYP3A4 in differentiated cells lysate via Elisa test
Cytochrome P450 3A4 (CYP3A4) BioAssay™ ELISA Kit (Mouse) utilizes the sandwich Enzyme Immunoassay technique
for in vitro quantative measurement of CYP3A4 in mouse, the microtiter plate provided in the detection kit has been pre-
coated with an antibody specific to CYP 3A4.Standers or samples are added to the appropriate microtiter plate wells with
a biotin-conjugated antibody specific to CYP3A4. Next, avidin conjugated to horseradish peroxidase (HRP) is added to
each microplate well and incubated. After TMB substrate solution is added, only those wells that contain CYP3A4, biotin-
conjugated antibody and enzyme-conjugated avidin will exhibit a change in color. The enzyme-substrate reaction is
terminated by the addition of sulfuric acid solution and the color change is measured spectrophotometrically at a wave
length of 450nm ± 10nm. The concentration of CYP3A4 in samples is then determined by comparing the OD of the
samples to the standard curve. The standard curve was drawn using curve expert 3.1 computer software by plotting on
the horizontal axis the cytochrome p450 concentrations of the standards and on the vertical axis the corresponding
average absorbance. To locate the concentration of cytochrome p450 in the samples, the average absorbance for each
sample on the vertical axis was located and the corresponding cytochrome p450 concentration was located on the
horizontal axis. (figure 1).
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Figure (1-2): Standard curves for mouse cytochrome p450
Figure (1): Standard curves for mouse cytochrome p450.
Results
Characterization ofBM- MSCsMSCs were isolated with perfect confluence form bone marrow flashing and cultured in
MEM medium containing 20% of FBS in primary culture then after passaging the cells were cultured in MEM mediumwith
10% FBS. Results showed attachment of isolated MSCs to the surface of culture falcon after 24 hrs of primary culture
while MSCs showed the spindle shape by after 80% confluence. Results showed the positive reactivity for CD 90 and
CD105 in which the cells stained withDAPI stain while the negative reactivity was noticed for both hematopoietic stem cell
markers CD34 and CD45 cells and stained with heamotoxilin stain Figure (2).
Figure (2): Immunocytochemistry analysis of isolated BM-MSCs: shows the positive reaction of cells towards
CD105 and CD90 markers of cell surface and the negative reaction for CD34 and CD45 cell surface markers.
Hepatocytes differentiation
during the differentiation experiment the cultured cells were monitored via inverted microscope for
morphological changes of MSCs during the stages of differentiation of hepatocytes Figure 3 (A,B,C,D,E and F).
CD 105
CD 90
CD 34
CD 45
0
0.2
0.4
0.6
0.8
1
1.2
020 40 60 80 100 120
O.D at 450 nm
Cytochrome oxidase p450 concentration in ng/ml
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Figure (3): Different stages of morphological patterns of hepatogenic differentiation of Mouse MSCs. A: the mesenchymal
stem cells isolated from mouse bone marrow; B: BM-MSCs under differentiation condition at day 10; C: BM-MSCs under
differentiation condition at day 16; D, E: Differentiated cells at day 19 and 21 F: cells at the end of differentiation
experiment.
Immunocytochemistry analysis of CYP3A4 of the differentiated cells
At the end of the differentiation experiments the resulting hepatocytes like cells were tested for Cytochrome oxidase p450
(CYP3A4 p450) expression, results showed that the majority of the differentiated cells (about 70% ) were stained with the
brown DAP stain (Figure 3-5 A), means these cells are positively reacted towards CYP3A4 p450 mouse anti human
primary antibody explain that the cells are positive for CYP3A4 enzyme expression, as well as some of these cells were
shown to be negatively reacted to the CYP3A4 p450 suggested that these cells may be undifferentiated cells or are not
completely differentiated to express the CYP3A4 p450 enzyme as a mature differentiated hepatocytes.
Figure (4): Immunocytochemical analysis of cytochrome oxidase p450 (CYP3A44 p450) in hepatocytes like cells. Positive
reaction in (A) in induced hepatocytes like cells, while (B) shows the negative reaction of cells against mouse antihuman
CYP3A4 p450.
Elisa assay of CYP3A4 in differentiated cells lysate
Results revealed that a significant increase in CYT3A4 concentration in hepatocyte lysate at 21 day (65.27± 0.52) ng/ml
in comparison with 14 and 7 33.0 ±0.0.72 and 16.73±0.92) ng/ml respectively (Figure 5).
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Figure (5): Level of CYT3A4 (ng/ml) in hepatocyte lysate after differentdifferentiation period.
ACKNOWLEDGMENTS
This study was supported by the Coordination of Iraqi center of Cancer researches and medical genetics and the college
of Science/department of biotechnology/Al-Nahrain Univ.
Discussion
The differentiation mechanism of mesenchymal stem cells is guided by a number of growth factors which affected the
expression of specific genes which directed the cells towards the cell type of interest in vitro. [9].Mesenchymal stem cells
(MSCs) have because of their plasticity and mulipotency had played a promising and reliable source for hepatic cell line
for the treatment of liver diseases [10].
Although the stem cell differentiation mechanism remains unclear to date, transdifferentiation might either be induced by
stimulating with suitable media/substrates/factors, or by genetic reprogramming in vitro [11]. Fibroblast growth factors
(FGFs), are involved at an initial stage of endodermal patterning to induce hepatic fate [9] Oncostatin M, a member of the
interleukin-6 cytokine family produced by hematopoietic cells, is required from the mid-fetal to the neonatal stages and
apparently coordinates liver development and hematopoiesis in the fetus [12]. This is consistent with the fact that FGF-4
may play a role in endoderm specification, and that HGF induces differentiation of hepatocytes that are not actively
proliferating [13]. Dexamethasone has been shown to have a specific differentiation-inducing effect on primary fetal
hepatic cells towards mature hepatocytes [14].
The cytochrome P450 (P450) superfamily consists of a large number of haem-containing mono-oxygenases that play a
pivotal role in the metabolism of many drugs and carcinogens [15]. Cytochrome P450 contain isoenzymes which catalyze
he metabolism of a wide number of drugs in the liver [16]. The non -hepatic cells may also possess this kind of catabolic
enzyme (CYp3A4) but in a ratio lower than in the hepatic cells in which these cells having a major role in drug metabolism
in the liver, this why the Elisa results showed a significant increase in the level of CYP3A4 at the end of the differentiation
protocol where the cells are fully differentiated in to hepatic cell line.
From all above we conclude that the bone marrow derived mesenchymal stem cells were able to differentiates into hepatic
cell line and expressed a higher level of the catabolic enzyme CYP3A4 which is involved in the detoxification processes of
many drugs achieved by the liver.
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