Developing a New Two-Step Protocol to Generate Functional Hepatocytes from Wharton's Jelly-Derived Mesenchymal Stem Cells under Hypoxic Condition

The shortage of donor livers and hepatocytes is a major limitation of liver transplantation. Thus, generation of hepatocyte-like cells may provide alternative choice for therapeutic applications. In this study, we developed a new method to establish hepatocytes from Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) cell lines named WJMSCs-SUT1 and WJMSCs-SUT2 under hypoxic condition. This new method could rapidly drive both WJ-MSCs cell lines into hepatic lineage within 18 days. The achievement of hepatogenic differentiation was confirmed by the characterization of both phenotypes and functions. More than 80% MSCs-derived hepatocyte-like cells (MSCDHCs) achieved functional hepatocytes including hepatic marker expressions both at gene and protein levels, glycogen storage, low-density lipoprotein uptake, urea production, and albumin secretion. This study highlights the establishment of new hepatogenic induction protocol under hypoxic condition in order to mimic hypoxic microenvironment in typical cell physiology. In conclusion, we present a simple, high-efficiency, and time saving protocol for the generation of functional hepatocyte-like cells from WJ-MSCs in hypoxic condition. The achievement of this method may overcome the limitation of donor hepatocytes and provides a new avenue for therapeutic value in cell-based therapy for life-threatening liver diseases, regenerative medicine, toxicity testing for pharmacological drug screening, and other medical related applications.


Introduction
Orthotopic liver transplantation has been shown to be an effective treatment for patients with end stage of liver dysfunction. However, this treatment is limited by the shortage of donor organs. Although hepatocytes transplantation has been shown to be successful treatment in some conditions such as liver-based metabolic disorders, the insufficient donor organs and hepatocytes remain obstacles for this technique [1]. Recently, stem cells are a promising tool for using as cell-based therapy because of their superior properties including self-renewal and broad differentiation potential into several cell types. To date, mesenchymal stem cells (MSCs) have been shown to obtain promising capacity not only multilineages differentiation potential but also immunomodulatory properties [2]. In addition, MSCs can be extensively expanded in vitro, due to effective cryopreservation and being easy to access from various sources such as bone marrow, adipose tissue, amniotic fluid, umbilical cord Wharton's jelly, and placenta [3][4][5][6]. These make MSCs become a good stem cell candidate for therapeutic purpose in clinical applications.
Umbilical cord Wharton's jelly is an enriched source of MSCs which has superior advantages over the other sources such as noninvasive collection, less ethical concern, and enrichment with MSCs [7]. Several studies have demonstrated that MSCs from various sources including Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) possess hepatogenic differentiation potential both in vitro and in vivo [8][9][10][11][12][13]. Interestingly, hepatocyte-like cells generated from adipose tissue-derived MSCs showed therapeutic effect on mice models with both acute liver failure and chronic liver 2 Stem Cells International injury [14,15]. Recent study also reported clinical improvement in patients with end-stage liver failure by chronic hepatitis C after transplantation with bone marrow-derived hepatocyte-like cells [16]. Based on these data, generation of hepatocyte-like cells from MSCs shows great potential in clinical use as regenerative medicine.
According to previous published protocols, several studies have shown expensive cost, time consuming, and multiple steps induction of MSCs into hepatic lineage [10,[17][18][19][20][21]. Therefore, a simpler method is needed for developing an effective protocol to generate functional hepatocyte-like cells from MSCs. In this study, we developed a new method to induce WJ-MSCs cell lines, WJMSCs-SUT1, and WJMSCs-SUT2, into hepatic lineage followed by characterization of the MSCs-derived hepatocyte-like cells (MSCDHCs) at both cellular and molecular levels. Here, we show the achievement of hepatogenic differentiation of WJ-MSCs by using our new induction protocol under hypoxic condition. The hepatocytelike cells generated from WJ-MSCs offer an alternative source of functional hepatocytes which will provide great advantages in liver disease treatments, drug discovery including toxicological research, and other medical applications.

Periodic Acid-Schiff (PAS) Staining.
After 4% paraformaldehyde fixation, cells were incubated for 5 minutes in 1% periodic acid (Sigma, St. Louis, MO, USA) and washed with distilled water prior to incubation with Schiff 's reagent (Sigma, St. Louis, MO, USA) for 15 minutes. After 5-minute wash in tap water, hematoxylin counterstain was performed for 1 minute. Cells were washed and visualized under light microscope (CKX41) (Olympus, Japan).
2.9. Statistical Analysis. All data were represented as mean ± standard deviation (SD) calculation. Statistical analysis was calculated by statistical software SPSS17.0 (SPSS Inc., Chicago, IL, USA) and results were analyzed by Student's t-test with significance at < 0.05.

Morphology of MSCs-Derived Hepatocyte-Like Cells (MSCDHCs).
Morphological changing of the cells was determined on day 0, 7, 10, and 18 following differentiation.
Upon induction, we observed morphological changing from fibroblastic cells of WJ-MSCs into polygonal round cells of hepatocytes feature. More than 80% of both WJMSCs-SUT1 and WJMSCs-SUT2 could be induced into hepatocytelike cells morphology at the end of induction. Moreover, WJMSCs-SUT1 changed morphology from fibroblastic shape into epithelial round shape earlier on day 10 of differentiation whereas WJMSCs-SUT2 showed this feature on day 12 ( Figure 2). For the control, both 2 cell lines did not show spontaneous differentiation into hepatocyte-like cells during the culture periods (data not shown). at different time points during differentiation periods. We found that undifferentiated stage of WJMSCs-SUT1 and WJMSCs-SUT2 could express low level of some hepaticspecific genes transcripts including AAT, ALB, CYP3A4, and G6P ( Figure 3). Upon induction, MSCDHCs generated from both 2 cell lines showed the significant upregulation of mature hepatic markers, AAT and CYP3A4, in a time-dependent manner. Additionally, we observed correlation between the expressions of key hepatic transcription factors, 4 , and mature hepatic marker ALB in MSCDHCs generated from both 2 cell populations. Interestingly, in late stage of differentiation, the WJMSCs-SUT2 had faster response to hepatic-lineage induction than WJMSCs-SUT1 did. This result was supported by the early expression of 4 transcripts on day 12 while it was undetectable in MSCDHCs from WJMSCs-SUT1 on the same day. However, both the WJMSCs-SUT1 and WJMSCs-SUT2 could express all tested hepatic-lineage markers at mRNA level at the end stage of differentiation period (Figure 3).

Expression of
We further analyzed more mature hepatic markers, albumin (ALB), and cytokeratin 18 (CK-18), at protein level. The low expression of CK-18 was observed in undifferentiated stage (day 0) of these 2 cell lines (Figures 4(i) and 4(m)). Following differentiation, more than 80% MSCDHCs generated from both 2 cell populations showed the significant upregulations of ALB (Figures 4(a)-4(h)) and CK-18 (Figures 4(i)-4(p)) in a time-dependent manner. These findings suggest that both WJMSCs-SUT1 and WJMSCs-SUT2 could also express hepatic-lineage markers at protein level in response to our induction medium in addition to the other hepatic mRNA expressions. Taken together, these results demonstrate that WJMSCs-SUT1 and WJMSCs-SUT2 could be induced into hepatocyte-like cells feature not only in a cellular phenotype aspect but also in hepatic marker expressions both at mRNA and protein levels.

Hepatocytes Functions of MSCs-Derived
Hepatocyte-Like Cells. The biological functions of hepatocytes were evaluated in the differentiated cells at various stages upon differentiation. First, the capacity of glycogen storage was analyzed by PAS staining. The results showed low level of PAS staining in undifferentiated stage (day 0) of WJMSCs-SUT1 and WJMSCs-SUT2. However, upon induction, MSCDHCs generated from both 2 cell lines displayed significant positive staining of glycogen granules in their cytoplasm as early as on day 7 and more stronger staining on day 12 and the strongest at day 18 ( Figure 5(a)). In addition, more than 80% of the differentiated cells from these 2 cell populations had capacity to accumulate low-density lipoprotein (LDL) inside the cells like functional hepatocytes in a time-dependent manner whereas undifferentiated cells (day 0) did not perform this ability ( Figure 5(b)). Furthermore, albumin secretion and urea production were also determined in order to confirm the hepatocytes functions. We found that undifferentiated stage (day 0) of WJMSCs-SUT1 and WJMSCs-SUT2 did not secrete detectable levels of albumin even though they have been detected albumin expression both at gene and protein levels. However, during differentiation processes, MSCDHCs WJMSCs-SUT2 generated from both 2 cell lines continuously secreted albumin since entry into maturation step from day 12 to day 18 ( Figure 6(a)). Finally, we also tested metabolic function of the differentiated cells to detoxify ammonia to urea which is a less toxic form. In consistency, the differentiated cells from WJMSCs-SUT1 and WJMSCs-SUT2 could produce urea in response to ammonia detoxification process at all points of differentiation period (Figure 6(b)). Taken together, these data suggest that both WJMSCs-SUT1 and WJMSCs-SUT2 could commit toward functional hepatocyte-like cells by our new hepatogenic induction protocol under hypoxic condition. Furthermore, the percentage of differentiated cells has been evaluated based on both hepatic phenotypes (ALBexpressing cells, CK-18-expressing cells) and functions (LDL and PAS positive stained cells) by counting the numbers of positive stained cells of each marker against the total numbers of the cells as references by their stained cell nuclei from the same sample. For hepatic phenotypes, the induced cells highly expressed hepatic markers both ALB (89.00 ± 1.00% for WJMSCs-SUT1, 88.33 ± 1.53% for WJMSCs-SUT2) and CK-18 (85.00 ± 2.00% for WJMSCs-SUT1, 83.00 ± 2.00% for WJMSCs-SUT2). For hepatic functions, these cells could highly store glycogen inside the cells as shown by positive stained cells of PAS staining (82.33 ± 2.52% for WJMSCs-SUT1, 87.00 ± 2.00% for WJMSCs-SUT2) whereas more than 80% of LDL positive stained cells were observed in both 2 cell lines. Taken together, our developed protocol under hypoxic condition could generate high yield of functional hepatocytes ranging from 80 to 90% of the induced cells from both WJMSCs-SUT1 and WJMSCs-SUT2. Here, we successfully generated a new method to establish high yield and functional hepatocyte-like cells from WJ-MSCs which may be advantages in clinical applications, hepatic toxicity drug screening test including toxicological research, and other medical applications.

Discussion
The WJ-MSCs are expected as a promising tool for therapeutic purposes. This is mainly due to their wide range differentiation capacity, noninvasive collection, and low immunogenicity. They have immunomodulatory as inhibitory effect on stimulated T cells in vitro that would be an advantage to allogenic transplantation [26]. Previous study found that human umbilical cord matrix stem cells could be successfully xenotransplanted into immunocompetent rats with little or no host immune response [27]. These findings support low immunogenic property of WJ-MSCs in vitro. In addition, WJ-MSCs have broader differentiation potential into several cell types such as insulin-producing cells [28], nerve cells [29], and cardiomyocytes [30] including hepatocyte-like cells [17]. Furthermore, preclinical studies found that WJ-MSCs Stem Cells International could improve the liver functions, restore the survival rate, and decrease liver fibrosis in animal model with liver injury [13,31]. Although WJ-MSCs have not been applied in clinical trials related to liver diseases, recent pilot study demonstrated the safety and successful treatment with placenta-derived MSCs in type 2 diabetes patients [32]. Based on their advantages, WJ-MSCs seem to possess beneficial effect on liver disease treatment.
In this study, we aim to investigate the efficiency of a new established method for in vitro induction of WJ-MSCs into hepatic lineage by testing with our human WJ-MSCs cell lines as WJMSCs-SUT1 and WJMSCs-SUT2. Based on several hepatogenic induction methods, we developed a new twostep protocol to induce in vitro hepatogenic differentiation of WJ-MSCs under hypoxic condition which resembles the normal microenvironment in human physiology. Typically, the existing published protocols that have been used for hepatogenic induction of MSCs are performed in normal atmosphere of 20% O 2 and 5% CO 2 . Here, we used hypoxic environment (5% O 2 and 5% CO 2 ) to accelerate hepatogenic differentiation potential of WJ-MSCs based on the superior advantages of this condition. In human ES cells, it has evidence that hypoxia is suitable for using in final hepatic induction to produce high yield of hepatocyte-like cells [33]. Moreover, hypoxia has been shown to activate hypoxia-inducible factor 1 (HIF-1) expression which has target gene asretinoic acid-receptor-related orphan receptor alpha ( ) transcripts in HepG2 [34]. The Rora upregulations are involved in controlling ADIPOQ and G6PC expressions which play roles in regulation of lipid and glucose metabolisms in hepatic cells, respectively [35]. From these data, hypoxia seems to provide appropriate environment 8 Stem Cells International  Figure 6: The capacities of the differentiated cells to secrete albumin and produce urea after induction for 18 days. Albumin secretion (a) and urea production (b) of MSCDHCs generated from WJMSCs-SUT1 and WJMSCs-SUT2 were determined by ELISA and colorimetric assay following the differentiation period, respectively. HepG2 was used as positive control. All data are presented as mean ±SD ( = 3). * < 0.05.
for maintaining physiological activities including hepaticlineage differentiation in liver cells. In addition, hypoxic preculturing of MSCs has been shown to increase cell homing, migration, and engraftment efficiency both in vitro and in vivo studies [36]. Therefore, it has possibility that hypoxic condition also enhances these efficiencies of MSCDHCs in addition to the improvement of the hepatogenic differentiation. The hepatocyte-like cells generated from hypoxic condition may retain these promising abilities which provide a great benefit in therapeutic purposes.
According to developing a new protocol, the achievement of MSCDHCs generation was observed from both WJMSCs-SUT1 and WJMSCs-SUT2 by sequential treatment with our new cytokines-based cocktail medium. Sequential exposure of these cytokines was mimicked from the behavior of mouse embryonic liver development. In early stage of differentiation, we used FGF-4, HGF, and nicotinamide as main induction factors. In facts, FGFs expressed by cardiac mesoderm play a role in induction ventral foregut endoderm to initiate early liver development. Afterward, liver bud formation is occurred and rapid hepatoblast proliferation is stimulated by HGF that is a crucial factor for hepatogenesis. Nicotinamide is a water-soluble amide form of niacin or vitamin B3 which serves as primary precursor of nicotinamide adenine dinucleotide (NAD + ) and the phosphorylated derivative NADP + synthesis [37]. Actually, NAD + and NADP + play roles in maintaining energy for cellular functions including DNA repair and genomic stability. For hepatogenic differentiation, nicotinamide has been shown to enhance the proliferation of primary rat hepatocytes and formation of small hepatocytes colonies [38]. Based on these data, we also used nicotinamide in combination with our first stage hepatogenic induction medium. In late stage of differentiation, we utilized OSM, dexamethasone, and ITS as major factors. It has been reported that OSM secreted by hematopoietic cells is involved in the maturation fate of fetal hepatocytes [18]. In addition to these factors, it has been reported that dexamethasone is required for maintaining the expression of liver-enriched transcription factors which are essential for stimulating liverspecific genes transcription [39]. ITS is known as chemically defined supplement for supportive in vitro proliferation in various mammalian cells. For hepatogenic induction, ITS seems to maintain cell survival as monolayer during the induction period. Thus, we also used dexamethasone and ITS to induce more mature hepatocytes differentiation in our system.
For hepatic-lineage marker expressions, 4 is a crucial liver-enriched transcription factor that plays an important role in stimulating the expression of liver-specific genes during liver development [40]. During the hepatogenic differentiation processes, early expression of 4 was detected in MSCDHCs generated from WJMSCs-SUT2 on day 12 which correlated with the upregulations of mature hepatic markers such as ALB, AAT, and CYP3A4. Similarly, MSCDHCs generated from WJMSCs-SUT1 showed the compatibility of 4 and hepatic-specific gene expressions but delayed to day 18. These findings imply that WJMSCs-SUT2 had a trend to differentiate into hepatic lineage in response to our induction method faster than WJMSCs-SUT1 did. Although these hepatocyte-like cells could express all mature hepatic markers, the expression of AFP which is a marker of immature hepatocytes was still detected even at late stage of differentiation. These data suggest that the hepatocyte-like cells or MSCDHCs generated from both 2 cell lines contain some hepatoblasts or hepatic progenitor cells. Similarly, previous studies demonstrated that human placenta-derived multipotent cells [41], umbilical cord matrix stem cells [17], and mesenchymal stromal cells derived from umbilical cord Wharton's jelly [10] were not yet fully differentiated into mature hepatocytes because AFP expression was still detected in the differentiated cells throughout the induction period. Based on these results, MSCs isolated from primitive origin seem to be reserved the immature phenotype even though they have driven toward specific lineage.
In a late stage of differentiation, MSCDHCs generated from both WJMSCs-SUT1 and WJMSCs-SUT2 achieved characteristics of functional hepatocytes which were confirmed by LDL uptake assay, PAS staining for glycogen storage, albumin secretion, and urea assay. In comparison to previous published protocols [10,[17][18][19][20][21], our method provided high yield production of hepatocyte-like cells from WJ-MSCs, approximately more than 80% of the induced cells. Our protocol does not need any extracellular matrix substances for coating the culture dish to enhance hepatogenic differentiation. Moreover, this method required a shorter differentiation period, approximately 2 weeks, as compared to the other protocols which performed longer, at least for 3 weeks. Here, we demonstrate a simple, high efficiency, and time saving protocol for the generation of functional hepatocyte-like cells from WJ-MSCs. Altogether, these findings provide a new alternative option for an in vitro hepatocytes generation which may overcome the limitation of liver cell donors for future use in clinical applications.

Conclusions
In summary, we report a simple, high efficiency, and time saving protocol for the generation of functional hepatocytelike cells from WJ-MSCs. This method succeeds to drive WJ-MSCs into hepatic lineage under hypoxic condition. The achievement of hepatogenic differentiation was confirmed by both phenotypes and functions including hepatic marker expressions both at gene and protein levels, glycogen storage, low-density lipoprotein uptake, urea production, and albumin secretion. The achievement of MSCDHCs generation from this new method may provide greater potential in clinical applications and overcome the shortage of donor livers which is a major limitation of orthotopic liver transplantation. The implications of this work are therapeutic value in cell-based therapy for liver disease treatments and other regenerative medicine, hepatic toxicity drug screening in pharmacological approaches, and other medical applications in the future.

Conflicts of Interests
The authors have declared that no conflict of interests exists.