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Cell Biology International (2008) 32, 959–965 (Printed in Great Britain)
Optimization of an effective directed differentiation medium for differentiating mouse bone marrow mesenchymal stem cells into hepatocytes in vitro
Xiao‑Lei Shia, Liang Maoa, Bi‑Yun Xub, Ting Xiea, Zhang‑Hua Zhua, Jun‑Hao Chenb, Lei Lib and Yi‑Tao Dinga*
aDepartment of Hepatobiliary Surgery, Affiliated Drum Tower Hospital, Medical College of Nanjing University, No. 321 Zhongshan Road, Nanjing 210008, PR China
bScientific Research Department, Affiliated Drum Tower Hospital, Medical College of Nanjing University, PR China


Abstract

We have used a uniform design to explore the most effective directed differentiation medium (MEDDM) for differentiating mouse bone marrow mesenchymal stem cells (mMSCs) into hepatocytes. Our methods involved arranging eight differentiation medium groups following uniform design. Flow cytometry was used to evaluate the percentage of ALB+ and CK18+ cells in each group. Factors and their concentrations in the MEDDMs were then identified. The MEDDMs were evaluated by their ability to differentiate mMSCs into hepatocytes by RNA and protein expressions and synthesis functions. FGF at 35ng/ml and OSM at 30ng/ml in the medium yielded the highest percentage of ALB+ and CK18+ cells. During directed differentiation using MEDDMs, ALB, CK18, TTR, AFP mRNAs were expressed. ALB and CK18 proteins were detected in the cells. The differentiated cells produced albumin and urea in a time dependent manner. Uniform design was adequate for choosing the MEDDM of mMSCs. MEDDM containing 35ng/ml FGF and 30ng/ml OSM was effective in differentiating mMSCs into hepatocytes.


Keywords: Bone marrow mesenchymal stem cells, Hepatocyte, Differentiation, Uniform design.

*Corresponding author. Tel.: +86 25 83304616x66866; fax: +86 25 83317016.


1 Introduction

Stem cells are cells with clonogenic and self-renewing capabilities which can differentiate into multiple cell types (Weissman, 2000). One particular stem cell in bone marrow has been shown to differentiate into hepatocytes (Theise et al., 2000a,b; Alison et al., 2000; Lagasse et al., 2000; Jian et al., 2002). The differentiation process of stem cells occurs in a specific microenvironment. Although complex, the process is initiated by adhesion-related events through signaling in the extracellular matrix, which contains soluble ligands controlling cell growth, differentiation and morphogenesis (Hemler and Rutishauser, 2000; Birchmeier and Rosenthal, 2000). Several cytokines, including fibroblast growth factor-4 (FGF-4), oncostatin M (OSM), hepatocyte growth factor (HGF) and epithermal growth factor (EGF), have been identified as controlling hepatic differentiation and maturation (Wells and Melton, 1999; Taga and Kishimoto, 1997; Somerset et al., 1997; Kamiya et al., 2001). Previously, we have successfully obtained hepatocyte-like cells through directed differentiation of mouse bone marrow mononuclear cells by using FGF-4, OSM, HGF and EGF as differentiation factors (Shi et al., 2005). However, the cytokines that affect the process of stem cell differentiation into hepatocytes in vitro have not been identified. In this paper, we identified and optimized the composition and concentration of the most effective differentiation medium. Using this medium, we demonstrate the differentiation of mouse bone marrow mesenchymal stem cell into hepatocytes using mRNA analysis, protein expression and cell functionality examination.

2 Materials and methods

2.1 Isolation and characterization of mouse bone marrow mesenchymal stem cells (mMSCs)

Bone marrow cells were prepared as previously described (Levite et al., 1991). Briefly, 5–7ml fresh bone marrow aspirate extracted from the tibias and the femora of C57BL/6 mice (Model Animal Research Center of Nanjing University, China) was suspended in Dulbecco's modified essential medium-low glucose (DMEM-LG) medium (Hyclone, Logan, UT, USA) and was centrifuged to pellet the cells and remove the fat. The cell pellet was re-suspended in DMEM-LG medium and fractionated on a density gradient generated by centrifugation of 1.077mg/ml percoll (Sigma–Aldrich, MO, USA) solution at 2200rpm for 30min at room temperature. Then the cells in the percoll interface were collected and rinsed twice. Cell viability was determined by the trypan blue exclusion test. Only suspensions with cell viability of more than >95% were used.

Bone marrow cells were inoculated at 5×105cells/cm2 in 10μg/ml fibronectin-coated (Sigma–Aldrich) culture flasks. The specific culture medium consisted of the following: 54% DMEM-LG, 36% MCDB-201 (R&D Systems Inc., Abingdon, UK), 10% fetal calf serum (FCS) (Sigma–Aldrich) with 1×insulin–transferrin–selenium (ITS), 10−8M dexamethasone, 10−4M ascorbic acid 2-phosphate, 100units/ml penicillin, 100μg/ml streptomycin (all from R&D Systems). The mMSCs cultures grew at 37°C in 5% CO2 in air. Three days later, the nonadherent cells were removed and fresh medium was added. Every four days, a medium change was performed. When the cultures reached 80% confluence they were trypsinized. After plastic adherence selection, mMSCs were cultured over four passages. The cells were harvested for cell surface characterization for identifying mMSCs.

Flow cytometry was used for analysis of cell surface molecules. Harvested P4 cells were washed with PBS containing 5% FBS. A total of 2×105 cells were suspended in 0.5ml of PBS containing 2% FBS, and incubated for 30min on ice in the dark with one or two of the following monoclonal antibodies: CD29-PE, CD44-PE, CD90-PE and CD45-FITC (Sigma–Aldrich). The labeled cells were analyzed on an FACSCalibur (Becton, Dickinson and Company, NJ, USA) by collecting a minimum of 10,000 events.

2.2 Selection of the most effective directed differentiation medium (MEDDM)

The uniform design method was adopted for the experiments, which involved four factors at four different levels. The four factors were hepatocyte growth factor (HGF, denoted by x1 (ng/ml)), fibroblast growth factor-4 (FGF-4, denoted by x2 (ng/ml)), epidermal growth factor (EGF, denoted by x3 (ng/ml)) and oncostatin M (OSM, denoted by x4 (ng/ml)). The four concentration levels were listed as follows. HGF: 0ng/ml, 20ng/ml, 40ng/ml, 60ng/ml; FGF-4: 0ng/ml, 20ng/ml, 40ng/ml, 60ng/ml; EGF: 0ng/ml, 10ng/ml, 20ng/ml, 30ng/ml; and OSM: 0ng/ml, 10ng/ml, 20ng/ml, 30ng/ml. Taking into consideration the interactions among different factors, while ignoring the high-order interactions, the uniform design system U8 (44) (http://www.math.hkbu.edu.hk/UniformDesign) was chosen for this experimental design.

Medium containing different compositions of the four differentiation factors (FGF-4, OSM, HGF and EGF) was designed using the uniform design system U8 (44). According to the U8 system, mMSCs were inoculated in eight different directed differentiation medium at 5×105cells/cm2 in 10μg/ml fibronectin-coated culture flasks. In addition to the following basic medium – 54% DMEM-LG, 36%MCDB-201, 10% FCS with 1×ITS, 10−8M dexamethasone, 10−4M ascorbic acid 2-phosphate, 100units/ml penicillin, 100μg/ml streptomycin – the differentiation groups were given different factors at various concentrations, as set out in Table 1. Cells were cultured in a humidified atmosphere of 5% CO2 and 95% air at 37°C. The medium was replaced every every days and cells were collected for further experiments after 21 days.


Table 1.

The uniform design experiment medium composition

GroupHGF (ng/ml)FGF-4 (ng/ml)EGF (ng/ml)OSM (ng/ml)
1400100
22040300
3020010
44002030
54040030
660603010
70601020
860203020


2.3 Flow cytometry analysis of ALB and CK18 positive cells

Cells at day 21 were detached from the flasks and stained for flow cytometry analysis. After immunofluorescent staining, cells were then fixed with 2% paraformaldehyde and left in solution until being analyzed with an FACSCalibur (Becton Dickinson). Primary antibodies included anti-mouse albumin (DakoCytomation, CA, USA) and anti-mouse CK18 (Pharmingen, CA, USA). Fluorescein (FITC) and phycoerythrin (PE)-conjugated antibodies (Sigma–Aldrich) were used as immunofluorescent secondary antibodies.

2.4 Directed differentiation of mMSCs with the MEDDM

The mMSCs were cultured in the MEDDM or the basic medium (as negative control) at 5×105cells/cm2 in 10μg/ml fibronectin-coated culture flasks for 21 days and cells were collected for subsequent experiments. Cell morphology was observed under an Olympus phase contrast microscope. Supernatants of the culture medium at days 0, 3, 6, 9, 12, 15, 18 and 21 were collected for albumin ELISA.

2.5 RT-PCR

Using trizol reagent (Sigma–Aldrich), the total RNA was extracted from fresh mMSCs, cultured mMSCs and directed differentiated mMSCs in the MEDDM, respectively. C57BL/6 mouse hepatocytes were used for positive control. cDNA was synthesized and amplified using a one-step RT-PCR kit (Clontech Laboratories, CA, USA). For albumin (ALB), cytokeratin18 (CK18), alpha fetal protein (AFP) and transthyretin (TTR), the following reaction conditions were used: reverse transcription at 50°C for 1h; denaturation of RNA/DNA hybrid and inactivation of reverse transcriptase at 94°C for 5min; PCR for 40 cycles; denaturation at 94°C for 30s; annealing at 60°C for 1min; extension at 72°C for 1min; and finally, extension at 72°C for 5min.

Primers used for amplification were as follows:

AFP (184bp): 5′-GTGAAACAGACTTCCTGGTCCT-3′, 5′-GCCCTACAGACCATGAAACAAG-3′;

Albumin (148bp): 5′-TCAACTGTCAGAGCAGAGAAGC-3′, 5′-AGACTGCCTTGTGTGGAAGACT-3′;

CK18 (127bp): 5′-TGGTACTCTCCTCAATCTGCTG-3′, 5′-CTCTGGATTGACTGTGGAAGTG-3′;

TTR (105bp): 5′-TCTCTCAATTCTGGGGGTTG-3′, 5′-TTTCACAGCCAACGACTCTG-3′;

β-Actin (587bp): 5′-CCAAGGCCAACCGCGAGAAGATGAC-3′, 5′-AGGGTACATGGTGGTGCCGCCAGAC-3′.

All primers were synthesized by Shanghai Bioengineer Company, China. mRNA levels were normalized to β-actin. The amplified products were subjected to electrophoresis in 1% agarose gels and stained with ethidium bromide.

2.6 Immunofluorescence

Regular culture mMSCs and differentiated mMSCs with the MEDDM were coated on glass slides and fixed with 4% paraformaldehyde for 10min at room temperature, followed by methanol for 2min at −20°C. Cells were then permeabilized with 0.1% Triton X-100 for 10min and the slides were blocked for 30min with a blocking and diluting solution before being incubated sequentially for 30min each with primary antibody (against mouse albumin, 1:100; Dako) and secondary antibody (FITC-conjugated, 1:100; Sigma–Aldrich). Between each steps, slides were washed with 10mM PBS in 1% BSA. Subsequently, the same slides were re-incubated with mouse CK18 antibody (1:100; Pharmingen) and TRITC-conjugated secondary antibody (1:100; Sigma–Aldrich), following the above protocol.

Mouse hepatocytes were used as positive controls and negative controls were designed by using PBS instead of primary antibody. Slides were observed with a Zeiss Axiovert fluorescence microscope (Carl Zeiss, Jena, Germany).

2.7 Albumin ELISA

Cell culture medium at various time points was collected as samples for albumin ELISA following standard protocol. Mouse hepatocytes were used as positive controls. Briefly, the ELISA plate wells were coated with the mouse albumin antibody (1:100, Dako) and incubated for 60min. Later, the wells were washed and blocked for 30min at room temperature. The wells were washed again. Assay samples or standards were then added to the wells for 60-min incubation. Afterwards, samples were removed and wells were washed. The secondary antibody HRP conjugate (1:10,000; Sigma–Aldrich) was then added to each well and incubated for 60min at room temperature. Finally, wells were washed and the substrate of TMB was added to each well for incubation (30min). TMB reaction was stopped by addition of 2M H2SO4 and the absorbance at 450nm was recorded using a microtiter plate reader.

2.8 Urea assay

Urea concentrations were measured with a colorimetric assay kit (Randox Life Sciences, Co. Antrim, UK). The mMSCs were plated at 5×105cells/cm2 on 1μg/ml fibronectin (FN)-coated six-well plates in MEDDM or regular culture medium. Cells (on days 3, 6, 9, 12, 15, 18, 21) were incubated in 2ml medium containing 5mM NH4Cl for 24h before the urea concentrations were measured. Mouse hepatocytes grown in monolayer with the same density were used as positive control and cell-free culture medium was used as negative control.

3 Results

3.1 Phenotypic analysis of mMSCs

The expression of different cell surface molecules, including CD29, CD44, CD45 and CD90, was determined by flow cytometry. For P4 cells, over 90% cells were CD45 negative, while CD29, CD44, CD90 were positive.

3.2 Optimization of the effective directed differentiation medium

Table 2 summarized the cell differentiation results of these medium as indicated by the percentage of cells expressing the liver-specific proteins, i.e., ALB (denoted by y1 (%)) and CK18 (denoted by y2 (%)).


Table 2.

Flow cytometry analysis of cells cultured in different media (mean ± SD)

GroupALB (%)CK18 (%)
147.76 ± 9.8419.85 ± 5.12
252.48 ± 10.1247.21 ± 8.64
378.95 ± 11.2369.54 ± 9.79
447.54 ± 8.7639.95 ± 8.22
585.91 ± 11.6767.37 ± 10.01
629.44 ± 5.6147.83 ± 6.91
750.88 ± 9.5344.74 ± 9.22
890.43 ± 10.7157.22 ± 9.87


For designing the composition of the medium, the following second-order polynomial model equation was used for the selection of variables:

(1)

The following equations were obtained from the studies:

(2)

(3) For model (2): F=15.39, P=0.0057, R2=68.73%, . For model (3): F=29.46, P=0.0013, R2=94.65%, , , . All these t-tests are significant at .

To apply differentiation factors to the regression models (2) and (3) for an optimum differentiation medium, the following equations were deducted:

(4) where was given by Eqs. (2) and (3) when , y1 reaches the maximum. To find the answer of , different levels of x2 were tested at every five unit and results suggested that x2 of 35ng/ml and x4 of 30ng/ml yielded the maximum y2.

According to the above statistical analysis, 35ng/ml FGF and 30ng/ml OSM were selected for the effective directed differentiation medium.

3.3 FGF-4 and OSM are effective enough to differentiate mMSCs into hepatocytes

3.3.1 Gene expressions of liver specific markers

To assess the directed differentiation of mMSCs into hepatic lineages, mRNA transcriptions of endodermal and liver specific genes, including ALB, CK18, TTR and AFP, were examined. Results suggested that all genes were transcribed in mMSCs cultured in the MEDDM, while they were non-detectable in either fresh mMSCs or regular culture mMSCs (Fig. 1).


Fig. 1

The mMSCs cultured in MEDDM expressed liver-specific genes on the mRNA level. (Lane 1: fresh mMSCs; lane 2: mMSCs cultured in MEDDM; lane 3: mMSCs cultured in regular medium; lane 4: mouse hepatocytes as positive control). RT-PCR was conducted as described in the Section 2, and the mRNA levels of different liver-specific genes were examined.


3.3.2 Protein expressions of liver specific markers

To further confirm the directed differentiation of mMSCs into hepatocytes, immunofluorescence was conducted to determine the expression of ALB and CK18 proteins. The results (Fig. 2) clearly demonstrate that ALB resides in the cytoplasm and cell membrane, while CK18 is scattered in the cytoplasm. ALB and CK18 expressions were also found to correlate with the degree of mMSCs differentiation. For example, on day 21, the ratio of ALB-positive cells reached 82.83±9.03%, and the ratio of CK18-positive cells reached 74.79±8.41%.


Fig. 2

Immunofluorescent staining of liver marker proteins (ALB-FITC, green; CK18-TRITC, orange under the same filter) in mMSCs cultured in the MEDDM (A) or regular medium (B) after 21 days. (1000×).


3.3.3 Hepatocyte functional activity

Albumin secretion, one characteristic of hepatocytes, was measured at various time points throughout cell differentiation. The mMSCs cultured in regular medium did not secrete any albumin, while mMSCs cultured in MEDDM produced albumin in a time-dependent manner. At day 15, the amount of albumin secretion reached its peak (Fig. 3).


Fig. 3

Albumin secretion of mMSCs was analyzed by albumin ELISA. Black circle symbols represented mMSCs cultured in the MEDDM. White circle symbols represented mMSCs cultured in regular medium, which was used as the negative control. Black triangle symbols represented primary mouse hepatocytes, which was used as the positive control.


To check hepatocyte functionality, urea production was examined. While mMSCs cultured in regular medium did not produce urea, cells cultured in the MEDDM produced urea after three days and the production of urea was found to be time-dependent (Fig. 4).


Fig. 4

Urea production of mMSCs was analyzed by urea assay. Black circle symbols represented mMSCs cultured in MEDDM. White circle symbols represented mMSCs cultured in regular medium, which was used as the negative control. Black triangle symbols represented primary mouse hepatocytes, which was used as the positive control.


4 Discussion

Liver development proceeds through multiple distinct steps with several growth factors and cytokines involved in each step in vivo. In mice, the initial event in liver development occurs at E9 and the foregut endoderm was directed to become the liver through interaction with the cardiogenic mesoderm (Zaret, 2000, 2001). During this process, growth factor FGF-4 is involved in endoderm specification and hepatic differentiation (Wells and Melton, 1999). Another factor, OSM, also plays an important role in hepatic maturation during the mid- to late-fetal stages (Taga and Kishimoto, 1997). In addition, HGF was reported to be an important component for liver development and differentiation, involved in the postnatal hepatic maturation as a paracrine factor (Somerset et al., 1997). Other cytokines such as EGF, insulin and TGF have also been reported to play crucial roles in liver development and regeneration (Kamiya et al., 2001).

Cell differentiation in vitro, however, may be different from the in vivo process. It is not known which cytokines affect the process of stem cells' differentiation into hepatocytes in vitro, though some researchers have suggested that the main players include HGF (Wang et al., 2004; Oh et al., 2000; Fiegel et al, 2003); HGF and EGF (Miyazaki et al., 2002); or HGF and FGF (Schwartz et al., 2002). Could all these factors – HGF, FGF-4, EGF and OSM (Wells and Melton, 1999; Taga and Kishimoto, 1997; Somerset et al., 1997; Kamiya et al., 2001; Zaret, 2000) – regulate the differentiation process in vitro? Do these factors interact with each other? This research addressed these questions and attempted to identify the most effective directed-differentiation medium through uniform design. Liver-specific mRNA, protein expression and cell functional assay were utilized for the validation of the MEDDM. Previous data from our lab clearly demonstrated hepatocyte-like cell generation when culturing mouse bone marrow mononuclear cells in a directed differentiation medium containing factors FGF-4, OSM, HGF and EGF (Shi et al., 2005). Based on previous results, new compositions of medium containing various amounts of HGF, FGF-4, EGF and OSM were set up for studies. Since ALB and CK18 are important markers of hepatocytes (Schwartz et al., 2002; Pan et al., 1998; Tateno and Yoshizato, 1996), they were chosen as two response variables for analyzing the relationship between the four factors (HGF, FGF-4, EGF and OSM) and variables.

Among the three common experimental designs (comprehensive, orthogonal and uniform), the uniform experimental design approach was selected. Though the comprehensive approach could be more accurate since all levels of all factors are chosen, the number of experiments could easily become unmanageable (Atkinson and Donev, 1992). For an experimental design of four factors and four levels, the number of experiments would be 256 every time if the comprehensive approach had been used. Orthogonal design utilizes a suit of specific orthogonal tables, and arrays the most representative experiments. Orthogonal design can reasonably decrease the number of experiments without compromising the information gained from the study. On the other hand, to avoid interaction effects (Goupy, 1993; Fang, 1980; Zeng, 1994), 66 experiments would have had to be run if the orthogonal design had been chosen. The uniform design of four factors and four levels, however, requires significantly less experiments. The key point for uniform design is to choose the most representative factors so as to obtain as much information as possible by a few experiments (Zeng, 1994; Fang et al., 2000). Uniform design approach was chosen because of the following advantages: (1) the number of experiments is less, thus it is economical; (2) it takes interaction effects into consideration; (3) regression models can be established to reveal the relationship between response elements and factors in the experiment domain; (4) experimental conditions can be optimized by identifying the optimal combination of factors at different levels; and (5) uniform design table is easily accessible for users on the web at http://www.math.hkbu.edu.hk/UniformDesign (Fang et al., 2000; Liang et al., 2001).

The U8 (44) system was chosen as the suitable uniform design of four factors and four levels. Accordingly, eight different mediums were set up to study the differentiation of mMSCs into hepatocytes. At the end of the differentiation period (day 21), flow cytometric analysis was conducted to evaluate the percentage of ALB and CK18 positive cells in each group. The stepwise regression analysis data suggested that the percentage of ALB-expressed cells correlated with OSM; the percentage of CK18-expressed cells correlated with both FGF-4 and OSM. These results are in accordance with the functions of different factors in liver development (Wells and Melton, 1999; Taga and Kishimoto, 1997; Zaret, 2000, 2001).

To explore the effect of the MEDDM, we identified the directed differentiated cells at gene level, protein level and cell's synthetical and metabolic function. We selected AFP, ALB, CK18 and TTR as markers of hepatocytes. AFP is a marker of endodermal differentiation as well as an early fetal hepatic marker. Expression of ALB starts in early fetal hepatocytes and reaches the maximal level in adult hepatocytes (Pan et al., 1998). CK18 is a cytoskeletal protein and expressed in mature hepatocytes. TTR represents endodermal differentiation and is expressed throughout liver maturation (Makover et al., 1989). We detected all these markers at the gene or protein level in the course of cell-directed differentiation. Cells cultured in the MEDDM also produced albumin, synthesized urea and glycogen in a time dependent manner. Although renal tubular epithelium cells and hepatocytes can synthesize urea, only hepatocytes can synthesize albumin (Dunn et al., 1991). Therefore, the combined data suggest that directed differentiated mMSCs through MEDDM have hepatocyte function. Furthermore, the differentiation efficiency of the new medium was much higher than previously reported (Shi et al., 2005). These data validated our experimental design approach and confirmed the effectiveness of the MEDDM containing FGF-4 and OSM in the differentiation of mMSCs into functional hepatocytes. Next, we will serve these functional hepatocytes as resources for bioartificial liver or hepatocyte transplantation.

The uniform experimental design approach was suitable for screening the most effective differentiation medium of mMSCs. The MEDDM containing 35ng/ml FGF and 30ng/ml OSM was effective in differentiating mMSCs into functional hepatocytes.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (No.: 30371391) and the National Natural Science Foundation of Jiangsu Province, PR China (No.: BK2003008).

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Received 25 October 2007/21 December 2007; accepted 2 April 2008

doi:10.1016/j.cellbi.2008.04.013


ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)