Efficient large-scale generation of functional hepatocytes from mouse embryonic stem cells grown in a rotating bioreactor with exogenous growth factors and hormones
- Shichang Zhang†1,
- Yunping Zhang†2, 3,
- Li Chen4,
- Tao Liu5,
- Yangxin Li2,
- Yingjie Wang1, 2Email author and
- Yongjian Geng2Email author
© Zhang et al.; licensee BioMed Central Ltd. 2013
Received: 23 April 2013
Accepted: 21 October 2013
Published: 2 December 2013
Embryonic stem (ES) cells are considered a potentially advantageous source of hepatocytes for both transplantation and the development of bioartificial livers. However, the efficient large-scale generation of functional hepatocytes from ES cells remains a major challenge, especially for those methods compatible with clinical applications.
In this study, we investigated whether a large number of functional hepatocytes can be differentiated from mouse ES (mES) cells using a simulated microgravity bioreactor. mES cells were cultured in a rotating bioreactor in the presence of exogenous growth factors and hormones to form embryoid bodies (EBs), which then differentiated into hepatocytes.
During the rotating culture, most of the EB-derived cells gradually showed the histologic characteristics of normal hepatocytes. More specifically, the expression of hepatic genes and proteins was detected at a higher level in the differentiated cells from the bioreactor culture than in cells from a static culture. On further growing, the EBs on tissue-culture plates, most of the EB-derived cells were found to display the morphologic features of hepatocytes, as well as albumin synthesis. In addition, the EB-derived cells grown in the rotating bioreactor exhibited higher levels of liver-specific functions, such as glycogen storage, cytochrome P450 activity, low-density lipoprotein, and indocyanine green uptake, than did differentiated cells grown in static culture. When the EB-derived cells from day-14 EBs and the cells’ culture supernatant were injected into nude mice, the transplanted cells were engrafted into the recipient livers.
Large quantities of high-quality hepatocytes can be generated from mES cells in a rotating bioreactor via EB formation. This system may be useful in the large-scale generation of hepatocytes for both cell transplantation and the development of bioartificial livers.
The management of patients with acute liver failure (ALF) is challenging. Emergency liver transplantation remains the most successful treatment in many cases of ALF. However, because of the shortage of available donor organs, only 20% of patients with ALF receive a transplant, and 80% die while on the waiting list . In the past decade, both hepatocyte transplantation and bioartificial livers have been investigated as a “bridge” or alternative to liver transplantation, which are promising treatment options for ALF patients awaiting a donor liver [2–4]. However, hepatocyte transplantation and the development of bioartificial livers entail a large quantity of high-quality hepatocytes, which also requires a donor liver. Thus an urgent need exists for an alternative and adequate supply of suitable hepatocytes for both hepatocyte transplantation and bioartificial livers .
Embryonic stem (ES) cells, pluripotent cells derived from the inner cell mass of preimplantation blastocysts, have the unique ability to give rise to all somatic cell lineages [6, 7]. In particular, the differentiation of hepatocytes or hepatocyte-like cells from ES cells in vitro and in vivo has been reported [8–10]. Such ES cell-derived hepatocytes are anticipated to be a useful source of cells for the treatment of liver diseases [11, 12]. However, none of the previous work with ES cells has achieved the efficient differentiation of ES cells into hepatocyte-like cells by using a protocol that meets the demands of the clinical applications.
Recent studies have demonstrated that a rotating cell-culture system (RCCS) can enhance the efficiency of human embryoid body (EB) formation and differentiation [13, 14]. The rotating bioreactor of the RCCS creates a simulated microgravity, allowing the ES cells to grow and differentiate in three-dimensional (3D) culture . Hepatocytes in 3D multicellular aggregates or spheroids have been shown to maintain their morphology and ultrastructural characteristics, resulting in higher degrees of liver-specific functions [16, 17]. Previous reports have also revealed that mouse ES (mES) cells differentiate into hepatocytes via embryoid body (EB) formation in vitro[18, 19].
In our prior study, we demonstrated that the growth and differentiation of ES cells in a biodegradable polymer scaffold within a rotating microgravity bioreactor yields organized functional hepatocytes that can be used in research on bioartificial livers and engineered liver tissue . Therefore, we hypothesized that a rotating bioreactor could be used for the efficient large-scale differentiation of ES cells into hepatocytes, which may further serve as an ideal source of cells for both hepatocyte transplantation and the creation of bioartificial livers. Here, to validate this hypothesis, we investigated whether mES cells can be induced to differentiate efficiently into cells with the morphologic, biologic, and functional characteristics of hepatocytes by using a rotating bioreactor and exogenous growth factors and hormones.
Materials and methods
ES cells culture
mES cells (CCE line) (Stemcell Technologies, Vancouver, BC, Canada) were cultured on gelatin-coated dishes in Iscove modified Dulbecco medium (IMDM) (Gibco, Grand Island, NY, USA) containing 15% fetal bovine serum (FBS) (Gibco), 0.1 mM nonessential amino acids, 2 mM L-glutamine, 0.1 mM monothioglycerol, 50 U/ml penicillin, 50 μg/ml streptomycin, and 1,000 U/ml recombinant mouse leukemia inhibitory factor (Chemicon International, Temecula, CA, USA).
EB formation and hepatic differentiation in a rotating bioreactor
mES cells were dissociated by using trypsin and seeded into a rotating bioreactor (Synthecon Inc., Houston, TX, USA) at a concentration of 1 × 106 cells/ml IMDM, which was supplemented with 10-7 M dexamethasone (Sigma, St. Louis, MO, USA), 1% dimethyl sulfoxide (DMSO) (Sigma), 20 ng/ml recombinant mouse hepatocyte growth factor (HGF) (R&D Systems, Minneapolis, MN, USA), 10 ng/ml recombinant human fibroblast growth factor-4 (FGF4) (Sigma), ITS (10 μg/ml insulin, 5 μg/ml transferrin, 5 ng/ml selenium) (Gibco), 15% FBS, 0.1 mM nonessential amino acids, 2 mM L-glutamine, 0.1 mM monothioglycerol, 50 U/ml penicillin, and 50 μg/ml streptomycin. The configuration of RCCS consists of four 50-ml-capacity bioreactors. The rotating bioreactor was set to rotate at a speed of 25 rpm at 37°C and with a humidified atmosphere containing 5% CO2, simulating microgravity to encourage cell 3D growth and differentiation. Every 3 to 4 days, 70% to 80% of the culture medium was replaced with fresh medium. In total, the culture duration was 21 days. mES cells were also differentiated by using the aforementioned exogenous growth factors and hormones in a culture plate, a two-dimensional culture (2D), as a control.
Visualization of cell growth and tissue processing
At various time points, EBs were removed from the rotating bioreactor and placed into six-well plates, and observed by using a stereomicroscope (SZX12; Olympus, Tokyo, Japan). Subsequently, the EBs were cultured and grown in six-well plates in the same differentiation medium described earlier and observed by using a phase-contrast microscope (IX50; Olympus). For histologic examination, the EBs were fixed for 24 hours in 10% neutral buffered formalin, routinely processed, and embedded in paraffin. Serial sections (4 μm) were then cut and stained with hematoxylin and eosin (H&E).
RNA extraction and RT-PCR
The total RNA was extracted by using an RNeasy Mini Kit (Qiagen, Valencia, CA, USA) and treated with RNase-free DNase (Qiagen). A reverse transcriptase-polymerase chain reaction (RT-PCR) was performed by using a Qiagen OneStep RT-PCR Kit with 10 U RNase inhibitor (Invitrogen, Carlsbad, CA, USA) and 20 ng of RNA, according to the manufacturer’s instructions. The PCR primers and the length of the amplified products were as follows: albumin (ALB) (5′-GCT ACG GCA CAG TGC TTG-3′, 5′-CAG GAT TGC AGA CAG ATA GT-3′; 55°C; 35 cycles; 260 bp); α-fetoprotein (AFP) (5′-TCG TAT TCC AAC AGG AGG-3′, 5′-AGG CTT TTG CTT CAC CAG-3′; 55°C; 35 cycles; 182 bp); tyrosine aminotransferase (TAT) (5′-ACC TTC AAT CCC ATC CGA-3′, 5′-TCC CGA CTG GAT AGG TAG-3′; 58°C; 35 cycles; 206 bp); glucose-6-phosphatase (G6P) (5′-CAG GAC TGG TTC ATC CTT-3′, 5′-GTT GCT GTA GTA GTC GGT-3′; 58°C; 35 cycles; 210 bp); transthyretin (TTR) (5′-CTC ACC ACA GAT GAG AAG-3′, 5′-GGC TGA GTC TCT CAA TTC-3′; 50°C; 35 cycles; 226 bp); and beta-actin (5′-TTC CTT CTT GGG TAT GGA AT-3′, 5′-GAG CAA TGA TCT TGA TCT TC-3′; 55°C; 35 cycles; 200 bp). The PCR products were separated by electrophoresis on 1.2% agarose gels and stained with ethidium bromide. Beta-actin was used as an endogenous control.
Western blot (WB) analysis
The protein concentration of the EBs lysates was measured by using a Bio-Rad Protein Assay Kit (Bio-Rad, Hercules, CA, USA). Approximately 20 μg of protein from each supernatant was subjected to 4% to 15% Tris–HCl gel electrophoresis (Bio-Rad) and then transferred onto a nitrocellulose membrane (Pierce, Rockford, IL, USA). The membranes were blocked in 2.5% nonfat milk in phosphate-buffered saline (PBS), followed by incubation with a goat anti-mouse ALB antibody (1:10,000) (MP Biomedicals, Aurora, OH, USA), a monoclonal anti-human/mouse AFP antibody (1:1,000) (R&D Systems), or a monoclonal anti-mouse cytokeratin 18 (CK-18) antibody (1:2,500) (PROGEN Biotechnik, Heidelberg, Germany). The blots were then incubated with a horseradish peroxidase (HRP)-conjugated donkey anti-goat IgG or goat anti-mouse IgG secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The protein bands were visualized by using enhanced chemiluminescence (Pierce), and images were captured by using medical x-ray film.
After deparaffinization and rehydration, the sections of the day-14 EBs were heated in a microwave at 100°C for 5 minutes in 10 mM sodium citrate for antigen retrieval, followed by blocking with 2% nonfat milk and 1% bovine serum albumin (BSA) in PBS for 45 minutes at room temperature. The slides were incubated with a goat anti-mouse ALB antibody (1:100) (Bethyl, Montgomery, TX, USA), a monoclonal anti-human/mouse AFP antibody (1:200) (R&D Systems), or a monoclonal against mouse CK18 antibody (1:500) (Progen Biotechnik) overnight at 4°C. The slides were then treated with biotinylated anti-mouse/rabbit IgG (Dako, Carpinteria, CA, USA) or biotinylated anti-goat IgG (Dako) for 60 minutes and followed by labeling with Texas Red-conjugated streptavidin (Jackson ImmunoResearch, West Grove, PA, USA) and DAPI (Sigma) nuclear staining. Immunofluorescence was examined with fluorescence microscopy.
For the immunocytochemistry analysis, day-14 EBs were plated on chamber slides and cultured for 5 days. Next, the EB-derived cells were fixed in 4% paraformaldehyde in PBS for 20 minutes at 4°C and permeabilized by using 0.1% Triton X-100 for 15 minutes. After blocking with a blocking solution, the cells were incubated with primary antibody, as described for the immunohistochemistry experiments, for 45 minutes at room temperature. The appropriate secondary antibody and Texas Red-conjugated streptavidin were then applied as described earlier.
ALB product of cultured EBs
At days 7, 14, and 21 of the rotating culture, EBs were seeded in 12-well plates at approximately 50 EBs/well and incubated with fresh medium for 72 hours in 5% CO2 at 37°C. The amount of ALB secreted into the medium was quantified by using a mouse ALB ELISA quantitation kit (Bethyl) under the conditions recommended by the manufacturer.
Cytochrome P450 activity assay
During the rotating culture, EBs were removed from the bioreactor at days 7, 14, and 21 and incubated with 10 μM pentoxyresorufin (Sigma) for 60 minutes at 37°C. The red fluorescence from the resorufin in the EB-derived cells, which was produced from the O-dealkylation of pentoxyresorufin by cytochrome P450, was visualized by confocal laser scanning microscopy (CLSM) (IX 71, Olympus) . The EBs were then immediately dissociated with trypsin, fixed in 4% paraformaldehyde in PBS, and analyzed by flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA). Additionally, EBs were seeded and statically cultured in six-well plates for 4 days, and the resultant EB-derived cells were also incubated with pentoxyresorufin and examined by CLSM .
Uptake of low-density lipoprotein (LDL)
EBs from day 7, 14, and 21 were incubated with 20 μg/ml DiI-Ac-LDL (Biomedical Technologies, Stoughton, MA, USA) for 4 hours at 37°C. The incorporation of the fluorochrome-labeled LDL into the EB-derived cells was visualized by CLSM. The flow-cytometric analysis  and CLSM observation of the DiI-Ac-LDL uptake by the EB-derived cells were performed in a similar manner to the assays described earlier for cytochrome P450 activity.
Periodic acid-Schiff (PAS) staining
PAS staining for intracellular glycogen was performed for 72 hours on both EBs sections and the EBs cultured in six-well plates by using a PAS staining kit (American Master Tech Scientific, Lodi, CA, USA) .
Indocyanine green (ICG) uptake
Rotating-culture EBs were removed from the bioreactor each week and further cultured in six-well plates for 72 hours. These EBs were then incubated in DMEM containing 0.1% ICG (Sigma) and 10% FBS for 30 minutes at 37°C. After rinsing 3 times with PBS, the cellular uptake of ICG was examined by both a stereomicroscopy and phase-contrast microscopy [25, 26].
Injection of EB-derived cells and culture supernatant
This study was approved by the Research Ethics Committee of the Southwest Hospital and Third Military Medical University. After 14 days of culture, the EBs were dissociated by using trypsin. These isolated cells were incubated with DiI-Cell-labeling Solution (Molecular Probes, Eugene, OR, USA) for 15 minutes at 37°C and then washed 5 times in PBS. The washed cells were resuspended in PBS at a concentration of 1 × 106 cells/ml, and the resultant cell suspension (0.2 ml) was transplanted into the spleens of 6- to 8-week-old nude mice under anesthesia. The cell-culture supernatant from the same EBs samples (0.2 ml) was injected into the spleens of nude mice as a control. Each experimental group consisted of six animals. One and 2 months after the cell transplantation and culture-supernatant injection, the nude mice were killed, and their organs, including the liver, spleen, lung, and kidney, were removed. The presence of tumors in these organs was macroscopically investigated. Samples of tissues were fixed in formalin, stained with H&E, and then microscopically confirmed. Other tissue samples were placed into OCT compound, and frozen sections 10 μm) were prepared and observed by fluorescence microscopy.
Statistical analyses were performed by using PRISM 5.01 (GraphPad Software). The data are presented as the mean values ± SD of n determinations, as indicated in the figure legends. ANOVA was used to calculate the significance of the differences between the mean values. A P value of < 0.05 was considered statistically significant.
EB formation and hepatic differentiation
Examination of the EBs under a stereomicroscopy revealed relatively homogeneous cell aggregates at day 7, with a few cystic EBs present (Figure 1C). Extensive cell clumps appeared after 2 to 3 weeks of suspension-culture. H&E staining clearly revealed the differentiation of the mES cells into cells with hepatocyte-like cells morphology. These cells exhibited polyhedral contours, large nuclei, and cordlike arrangement within the EBs at days 14 and 21 (Figure 1E, F), but not at day 7 (Figure 1D).
Further to confirm the hepatic differentiation of the ES cells in the EBs, we seeded the EBs in six-well plates and cultured the cells in the same differentiation medium (Figure 1G). Morphologically distinct cell types were observed in subculture. In particular, many of the day-21 EB-derived cells were characterized by a uniform pyramidal shape, large round nuclei, and dark granular deposits within the cytoplasm after 3 days (Figure 1H) and 5 days (Figure 1I) of culture, consistent with our histologic observations.
Hepatic marker expression
Immunohistochemical and immunocytochemical staining
Metabolic enzyme activity
Glycogen and ICG staining
Transplantation of EB-derived cells
Many hepatic differentiation protocols involve EB formation from ES cells grown in static culture [25, 27]. Similar to embryos, EBs contain an endoderm, ectoderm, and mesoderm, which are important for the induction of complex differentiation . Several techniques, such as the hanging-drop and conical-tube methods , have been developed to facilitate EB generation. The stirred-tank bioreactor and perfused 3D bioreactor have been applied for the hepatic differentiation of ES cells [30, 31]. However, none of the current methods is sufficiently stable and controllable for the large-scale differentiation of ES cells for practical applications. Here, we used a rotating bioreactor to induce EB formation and hepatic differentiation. Cell culture in a rotating bioreactor has been shown to support the efficient, large-scale production of transplantable cells for bone-tissue engineering and of hematopoietic cells [32, 33]. To the best of our knowledge, although a few studies have examined the formation and multiple-tissue or tissue-specific differentiation of EBs in a RCCS [15, 34, 35], no one has reported the hepatic differentiation of mES cells via EB formation in a rotating bioreactor. In our previous study , the hepatic differentiation of mES cells within the 3D condition of a rotating bioreactor has been described, which is similar to the present study. A 3D-culture system consisted of biodegradable scaffolds, growth-factor-reduced Matrigel, and a rotating bioreactor in our previous study. Scaffolds and Matrigel provided a 3D environment for the cell growth, proliferation, and differentiation of mES cells. Tissue mass with functional hepatocytes was obtained from the rotating bioreactor for bioartificial livers and engineered liver tissue. In this study, however, the formation of EBs was dependent on mES cell self-assembly. A native 3D microenvironment with cell-cell and cell-matrix interactions was recapitulated in EBs. A large number of EBs with functional hepatocytes were prepared in the rotating bioreactor. This method is suitable to large-scale generation of hepatocytes derived from ES cells for cell transplantation and the development of bioartificial livers. Therefore, our data indicate that the rotating bioreactor is an effective tool to provide a native 3D microenvironment for the large-scale production of hepatocytes derived from ES cells.
Several studies demonstrated that the optimal speed for EB formation is 15 to 20 rpm in the rotating bioreactor [33, 36]. To enhance the efficiency of EB formation, we chose a slow rotational speed and a high cell concentration, resulting in large numbers of homogeneous EBs within 1 week. By culturing ES cells in suspension in this dynamic and mild environment, which is characterized by extremely low fluid shear stress and optimal nutrient and gas exchange, our protocol for EB formation not only avoids massive cellular damage but also controls the extent of EB agglomeration, thus reducing large central necrosis in EBs. More important, both histologic and morphologic observations revealed that this setup is more beneficial for the differentiation of ES cells into hepatocytes than standard conditions.
It has been previously shown that exogenous growth factors and hormones play an important role in the differentiation of ES cells into hepatocytes and in the expression of the hepatocyte phenotype . However, it is still unclear which growth factor, hormone, or combination of the two is critical for the hepatic differentiation of ES cells. Additionally, the optimal timing for the use of these factors and hormones to differentiate ES cells into hepatocytes is not known. In the current study, we found that the hepatic differentiation of ES cells in a rotating bioreactor was more efficient with than without the presence of HGF, FGF4, dexamethasone, DMSO, and ITS. More specifically, the early use of medium containing these factors resulted in the efficient differentiation of ES cells into hepatocytes, as determined by morphologic analysis (data not shown).
In this study, we investigated the expression of TTR, AFP, ALB, G6P, and TAT at the mRNA level. Among these genes, TTR is a marker of endodermal differentiation, AFP represents endodermal differentiation as well as the early differentiation of fetal hepatocytes , ALB appears in early fetal hepatocytes and reaches a maximal level in adult hepatocytes , G6P is predominantly expressed in hepatocytes in the late gestational and perinatal stages , and TAT is an excellent enzymatic marker for perinatal or postnatal hepatocyte-specific differentiation and represents an important function of the liver . In general, the expression of these specific genes in cells derived from EBs is characteristic of differentiated hepatocyte-like cells, although a proportion of these genes is also expressed in the yolk sac and other cells. The expression of such genes in EBs differentiating in the presence of growth factors parallels the expression observed in the embryonic liver in vivo. However, in our tests, the mRNAs of TTR, AFP, ALB, G6P, and TAT were expressed by day 7, and the expression of most of the genes, such as ALB, G6P, and TAT, was noticeable earlier than in previous reports [27, 41]. The level expression of these genes was higher in the EB-derived cells cultured in the bioreactor than the mES cells differentiated in the 2D culture. Because the expression of the ALB, AFP, and CK18 proteins was also detected in the day-7 EBs by WB analysis, and based on our immunostaining results, we believe that growth of ES cells in a rotating bioreactor supplemented with exogenous growth factors not only strongly promotes differentiation into hepatocyte-like cells but also enhances cell maturation.
The assay for ALB synthesis is well known as a relatively specific test for the presence and activity of hepatocytes during the hepatic differentiation of stem cells [23, 25]. We examined the ALB synthesis of mES cell-derived EBs to confirm the differentiation of the ES cells into functional hepatocytes. However, we failed to detect ALB directly at measurable levels during the rotating bioreactor culture period. As the probable reason for this issue was the relatively low cell concentration in the bioreactor, we removed the EBs from the bioreactor and seeded them in 12-well plates at a concentration of approximately 50 EBs/well. After incubation with fresh medium for 72 hours, these EBs secreted ALB into the culture medium. The ability of the EBs to synthesize ALB at days 14 and 21 was approximately sixfold to eightfold higher than at day 7, confirming the hepatic differentiation of the EBs.
Because normal hepatocytes possess many complex functions, in addition to our ALB analysis, we used other independent functional assays to verify further the hepatocyte-like attributes of the differentiated ES cells. Four functional tests were performed on both the EBs from the rotating culture and the EB-derived cells from the static culture more precisely to characterize the hepatic functions of the differentiated cells. Our data demonstrate that the EB-derived cells possessed cytochrome P450 activity, stored glycogen, and took up ICG and LDL, which were higher than that of cells derived from 2D culture. Together, all of these results strongly suggest that the cells differentiated in the rotating bioreactor, along with growth factors and hormones, were functional hepatocytes.
The use of a rotating bioreactor for the differentiation of functional hepatocytes from ES cells, as reported in this study, has two advantages. First, the hepatic differentiation of ES cells in a rotating bioreactor is more effective than in traditional 2D culture. Second, large quantities of differentiated cells can be generated by using this technique. In the present study, most of the differentiated cells exhibited the complex functions of mature hepatocytes and the 3D characteristics of multicellular aggregates, conforming to the requirements of high-quality, high-density hepatocyte cultures for both hepatocyte transplantation and the seeding of bioartificial liver devices. Further research is needed to investigate the therapeutic potential of the differentiated cells grown under these conditions and to optimize the protocol for the differentiation of the EBs to avoid necrosis.
Acute liver failure
Bovine serum albumin
Confocal laser scanning microscopy
Fetal bovine serum
Fibroblast growth factor-4
Hematoxylin and eosin
Hepatocyte growth factor
Iscove modified Dulbecco medium
Rotating cell-culture system
This research was supported by the National Natural Science Foundation of China (31070880, 81000182). We thank Corina Rosales for help with cell culture and Hua Lu for skillful assistance with the immunofluorescence experiments.
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