Ameliorating effect of encapsulated hepatocyte-like cells derived from umbilical cord in high mannuronic alginate scaffolds on acute liver failure in rats.

OBJECTIVES: In this study, effects of encapsulated umbilical cord stem cells (UCSCs)-derived hepatocyte-like cells (HLCs) in high mannuronic alginate scaffolds was investigated on CCl4-induced acute liver failure (ALF) in rats.
MATERIAL AND METHODS: UCSCs were encapsulated in high mannuronic alginate scaffolds. Then the UCSCs differentiated into HLCs for treatment of CCl4-induced ALF in rats. Thirty rats randomly divided into 5 groups: Intoxicated group received only CCl4 to induce ALF. In other groups including cell-free, UCSCs and HLCs, alginate scaffolds were transplanted into the liver 4 days after CCl4 injection. Biochemical markers including albumin (ALB), blood urea nitrogen (BUN), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) were evaluated. Histological changes and gene expression of ALB, alpha-fetoprotein (AFP), and cytokeratin 18 (CK-18) were also assessed.
RESULTS: Expression of CK-18 significantly increased in HLCs compared to the UCSCs in vitro. This indicates that UCSCs can effectively differentiate into the HLCs. In CCl4-intoxicated group, BUN, AST and ALT levels, and histological criteria, such as infiltration of inflammatory cells, accumulation of reticulocytes, nuclear pyknosis of hepatocyte and sinusoidal dilation, significantly increased. In this group, ALB secretion significantly decreased, while AFP expression significantly increased. Both UCSCs and HLCs encapsulated in alginate scaffolds effectively attenuated biochemical tests, improved liver cytoarchitecture, increased expression of ALB and reduced AFP expression.
CONCLUSION: Finding of the present study indicated that encapsulation of UCSCs or HLCs in alginate mannuronic scaffolds effectively improve CCl4-induced ALF.

Introduction

Acute liver failure (ALF) is a rare but sudden clinical syndrome with high rate of mortality, which arises from a fast and extensive hepatocellular necrosis (1). Liver transplantation is the only choice for treatment of ALF. However, scarcity of human donors has strongly limited the transplantation of the liver (2). Currently, regenerative medicine has opened a window for treatment of the end-stage hepatic diseases (3). Tissue engineering has been introduced by using living cells on a suitable substrate or scaffold to restore, maintain, or enhance tissue and organ function (4).

Mesenchymal stem cells (MSCs) are attractive sources for cell therapy. They can be easily isolated from various tissues including bone marrow, adipose tissue and umbilical cord (5). Umbilical cord mesenchymal stem cells (UCSCs) have unique immunosuppressive properties enabling them to evade host rejection (6).

In liver tissue engineering, in order to MSCs differentiation into mature hepatocytes in vitro, there are different methods and growth factors to provide stimuli and maintain cellular function (7).

A three-dimensional (3D) biocompatible scaffold provides a supporting framework for cell growth, cell-cell or cell-matrix interactions, cell adhesion and cell differentiation (8).

It has been revealed that 3D culture of hepatocytes keeps their functions better than their culturing in two-dimensional (2D) environment. Aleahmad et al. demonstrated that heparin collagen 3D scaffold enhanced hepatocyte differentiation of UCMSCs, and a hepatocyte cell line culture in 3D condition was shown to increase gene expression pattern and metabolic activity compared to 2D environment. 3D culture also improved the expression of hepatic nuclear factor-4, the key regulator of hepatocyte differentiation, in hepatocytes-like cells (HLCs) derived from human Wharton’s jelly MSCs (WJ-MSC). This evidence demonstrated that 3D culture can enhance the hepatocyte function loss and improve the hepatocyte differentiation efficiency (9).

Alginate has been considered as a suitable scaffold that plays a critical role in tissue engineering and enhances the function of mature hepatocytes and maintains mature hepatocyte function within bio-artificial livers (10). Alginate is an unbranched natural copolymer composed of D-mannuronate and L-guluronate, linked together by 1, 4 bonds. Due to its relative stability, biocompatibility, adjustable porosity and simplicity of use, alginate is thus a biomaterial of choice to cell therapy (11).

It has been demonstrated that high mannuronic alginate effectively enhanced albumin (ALB) secretion in encapsulated hepatocytes compared to the high guluronic content encapsulation (12).

Until now, the ideal marker to trace the pathway of stem cell development does not exist, but under all circumstances, alpha-fetoprotein (AFP) is a very promising candidate for the study of differentiation or retrodifferentiation by virtue of its strong correlation with fetal gene expression. In addition, AFP is a biological marker, which increases in liver injuries (13).

The expression of ALB that is the abundant protein synthesized by mature hepatocytes starts in early fetal hepatocytes and reaches the maximal level in adult hepatocytes. Cytokeratin 18 (CK-18), a cytoskeletal protein, is expressed in mature hepatocytes. Hence, expression level of these genes can clarify the statue of liver function (10).

Several protocols for generating functional hepatocytes have been reported in literature; however, reproducible and efficient differentiation remained challenging under conventional (2D) culture. In this study, we used a 3D culture condition for generating functional HLCs from UCSCs.

Most of the transplantation sites, such as intraperitoneal or subcutaneous, are unable to maintain cell viability for long time periods as the transplanted cells have poor access to oxygen and nutrients (14). The present study was conducted to investigate whether the transplantation of mannuronic-rich alginate beads encapsulated UCSCs-derived HLCs into the liver could ameliorate ALF induced by carbon tetrachloride (CCl4) in rats while cell-scaffold constructs were implanted directly into the liver between the lobes.

Materials and Methods

Fresh human umbilical cords were collected aseptically from the mothers undergoing uncomplicated elective cesarean section after receiving their written consent. The umbilical cords dissected to earn small pieces of Wartons’ jelly. The Wartons’ jelly pieces were cultured as explants in DMEM (low glucose) with 20% fetal bovine serum.

The explants were left undisturbed for 3 days in CO2 incubator at 37 °C. After 10 days, the explants were removed from flasks. After 5 days, the flasks were passaged and the isolated cells were used in the experiment (15). The isolated cells were also characterized using flowcytometry and assessment of differentiation potential.

After trypsinization, 5 × 105 cells were resuspended in PBS and 5% FBS and incubated with phycoerythrin (PE)- or fluorescein isothiocyanate (FITC)-conjugated antibodies against mesenchymal cell surface markers, CD73 and hematopoietic markers, CD31 and IgG (ebio science, USA) for 30 min at 4°C. Labeled cells were assayed by Dako Glaxy flowcytometer. Mouse isotype-matched antibody was used as controls (ebio science, USA).

UCSCs were differentiated toward the adipogenic and osteogenic linage according to the protocol described by Hashemitabar et al (15).

A low viscosity, high mannuronic acid alginate solution (2% wt/vol, Nova Matrix, Philadelphia, PA) was prepared. Briefly, the alginate powder was dissolved in buffer solution containing 0.15 M NaCl and 0.025 M HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (16). The isolated UCSCs at a density of 3×106 cells/ml were resuspended in 3 ml of the aforementioned solution and dropped into a 102 mM CaCl2 solution and allowed to be polymerized for 20 min. Then, alginate scaffolds containing UCSCs were rinsed 3 times in 0.15 M NaCl. The constructs were finally cultured in 25-cm2 T-flasks. Differentiation of UCSCs into HLCs was induced using a 4-step protocol as follows (17):

Step I: DMEM (low glucose) and 10 ng/ml fibroblast growth factor (FGF-4) for 2 days.

Step II: DMEM (low glucose), 1% insulin-transferrin-selenium (ITS), and 20 ng/ml hepatocyte growth factor (HGF) for 2 days.

Step III: DMEM (low glucose), 100 nM dexamethasone, 1% ITS, 10−7 mg glucagon, 10 ng/ml oncostatin M (OSM) and 20 ng/ml HGF for 2 days.

Step IV: Step III medium and 100 nM trichostatin A (TSA) or 1% dimethyl sulfoxide (DMSO) for 14 days. The medium was exchanged every 2 days (17).

The alginate free-cell scaffolds, UCSCs scaffolds and HLCs scaffolds were assessed by a scanning electron microscope (SEM) before transplantation. Briefly, the scaffolds on day 14 were washed twice with cold PBS and fixed by 2.5% glutaraldehyde for 24 hr. The scaffolds were incubated with 2.5% glutaraldehyde buffered in 0.15 mol/l sodium cacodylate at 20˚C for 1 hr (pH=7.2). After fixation, the scaffolds were repeatedly rinsed in cacodylate buffer. The cultures were then dehydrated in a graded series of ethanol (50, 70, 95 and 100% alcohol). Then, chemical drying was achieved by using ethanol/Hexamethyldisilazane (HMDS, Merck, Germany) mixture and pure HMDS. The preparations were sputter-coated with gold-palladium before SEM (18). Porosity of the scaffolds was determined by the free ImageJ software. (http://imagej.nih.gov/ij/index.html)

The study protocol was approved by the Ethics Committee of Ahvaz Jundishapour University of Medical Sciences (approve number: IR. Ajums. REC. 1394. 605).

Thirty rats (Albino, male, 250 to 300 g) were obtained from Experimental Animals Research Center of Ahvaz Jundishapur University of Medical Sciences and housed for 1 week in the animal room of the Department of Anatomical Sciences for adaptation to the environmental before the experiments. The experimental room had a controlled temperature at 20± 5 °C with 12 hr light/dark cycles. The animals had access to food and water freely.

All surgeries were performed under deep anesthesia and sterile surgical techniques. Animals were anesthetized with 10% ketamine and 2% xylazine (Alfasan, Woerden, Netherlands) through intraperitoneal injection and then fixed in supine position. The surgical area was disinfected with iodophore. A midline abdominal incision (2 to 3 cm) was made and alginate scaffolds were placed between the lobes of the liver (The 15 alginate sheets, with a diameter 6 mm and height 1 mm). Then, the abdominal incision was sutured in two layers with 5-0 absorbable suture. Each rat was maintained in a separate cage and checked daily for their health status. To induce ALF, CCl4 at the dose of 400 μl/kg was peritonealy injected for two consecutive days. CCl4 was dissolved in olive oil (1:1) (19).

The rats were randomly divided into 5 groups:

1- Negative control group: received olive oil via intrapritoneal injection.

2- CCl4-intoxicated group: received only CCl4 via intrapritoneal injection.

3- Cell-free alginate transplanted group: cell-free scaffolds were transplanted into the liver 4 days after administration of CCl4.

4- UCSCs transplanted group: UCSCs alginate scaffolds equivalent to one million cells were transplanted into the liver 4 days after administration of CCl4.

5- HLCs alginate beads transplanted group: HLCs alginate scaffolds equivalent to one million cells were transplanted into the liver 4 days after administration of CCl4.

Fourteen days after transplantation, the rats were euthanized, the liver tissues removed and fixed in 10% neutral formalin solution for histological assessments (20).

Blood samples were collected and centrifuged for 15 min at 10,000 g before and after the beginning of the experiments. The supernatant was stored at -80 °C until use. ALB, blood urea nitrogen (BUN), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) were determined using photometric kits (Pars Azmun, Tehran, Iran) according to the manufacturer’s instruction.

After blood collection, the rat livers were fixed in 10% formalin solution. Paraffin sections of 4-6 µm were prepared and stained with hematoxylin and eosin (H&E). Six microscopy slides per animal were examined for assessment of histological changes such as congestion of erythrocytes, infiltration of inflammatory cells, nuclear pyknosis and sinusoidal dilation. For assessment of nuclear pyknosis, the average percentage of nuclear pyknosis was determined by dividing the number of nuclei with the pyknosis in a randomly microscopic field by the total number of nuclei in the same field and the result multiplied by 100. Infiltration of inflammatory cells, sinusoidal dilation and congestion of RBCs were graded into 4 categories: normal (0), weak (1), moderate (2) or intense (3) and the averages were considered. For each slide, the mean of 6 fields was calculated. Slides were read in a “blind” fashion (21).

Real-time RT-PCR was performed on day 14 after transplantation to determine the mRNA expression levels of ALB, AFP, and CK-18. The RNA was isolated from the liver tissue by using the RNeasy plus Mini Kit (Qiagen, Gaithersburg, MD, USA) according to the manufacturer’s instructions. The extracted RNAs were quantified using a nanodrop spectrophotometer (Thermo Scientific NanoDrop 2000c Spectrophotometer, USA).

The cDNA was synthesized using a QuantiTect Reverse Transcription Kit (Qiagen, Gaithersburg, MD, USA).

The primer sequences used in this study were ALB: 5-GCCTGCTGACTTGCCTTCATTAG-3 (forward primer) and 5- TCAGCAGCAGCACGACAGAGTA-3 (reverse primer);

AFP: 5-GAAACCCACTGGAGATGAACAGTC-3 (forward primer) and 5-AAGTGGGATCGATGCAGGA-3 (reverse primer);

CK-18: 5-GATCGACCTGGACTCCATGAGAA-3 (forward primer) and 5-CCGTTGAGCTGCTCCATCTGTA-3 (reverse primer);

GAPDH: were 5-TGCTGGTGCTGAGTATGTCGTG-3 (forward primer) and 5-CGGAGATGATGACCCTTTTGG-3 (reverse primer).

PCR amplification was performed over 40 cycles using a SYBR Green PCR Master Mix (Takara, Beijing, china) with the following program: 95 ˚C for 10 min, 95 ˚C for 15 sec, 5 ˚C for 45 sec and 60 ˚C for 40 sec. Data were analyzed using the 2-ΔΔCT method. Expression values were corrected for the housekeeping genes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (22).

Data were presented as mean ± standard deviation and were statistically analyzed using SPSS software (Version 22, Chicago, Illinois, USA) by one-way analysis of variance (ANOVA). The difference between the means was considered significant when P was < 0.05.

Results

Flowcytometric analysis indicated high expression level of CD 73 (88.9%), while low expression of CD31 (0.12%) was observed in the UCSCs.

The cells treated with adipogenic induction medium for 21 days showed numerous lipid vacuoles by using Oil Red O staining. In osteogenic induction medium, deposition of calcium showed red color by Alizarin Red stating. In non-treated control cells, no mineralized matrix and lipid vacuole were observed (Figure 1).

Figure 1

Differentiation of UCSC into adipocytes (right) and osteoblasts (left) have been shown

UCSCs had fibroblast like shape. HLCs were close together and formed cell clusters. Inverted microscopy reveled cell clusters that we call them hepatospher, since they had clear cytoplasm and nuclei.

In the SEM images, the cell-free porous scaffolds had numerous interconnected pores. The scaffolds had porosity with mean pore size of 176.88±0.25 SEM evaluations showed that UCSCs and HLCs formed continuous sheets of cells. The morphology of encapsulated UCSCs was flat and elongated. While, the encapsulated HLCs showed polyhedral morphology with confluence similar to the hepatic-like clusters (Figure 2).

Figure 2

Photomicrograph of scanning electron microscope (SEM) staining. A, cell-free alginate bead; B, umbilical cord stem cells (UCSCs) alginate bead; C, hepatocyte-like cells (HLCs) alginate bead

In CCl4-intoxicated group, AST, ALT, ALP and BUN levels significantly increased and ALB concentration significantly decreased compared to negative control and both cell-treated groups (P<0.05). Transplantation of cell-free scaffolds had no influence on the concentration of the biochemical markers. Transplantation of both encapsulated UCSCs and HLC induced a significant decrease in AST, ALT, ALP and BUN levels and significantly increased ALB secretion (P<0.05). The levels of the biomarkers and ALB were more pronounced in HLC transplantation in comparison with the UCSCs transplanted animals (P<0.05). (Figures 3 and 4).

Figure 3

Plasma levels of biochemical markers in control and experimental treated groups. Values expressed as mean±SD for 6 rats. *P<0.05, **P<0.01, ***P<0.001, # P<0.05; * and # symbols respectively indicate comparison to the control and CCl4-intoxicated groups. UCSCs: Umbilical cord stem cells, HLCs: Hepatocyte-like cells

Figure 4

Albumin (ALB) secretion in control and experimental treated groups. Values expressed as mean±SD for 6 rats. *P<0.05, **P<0.01, # P<0.05; * and # symbols respectively indicate comparison to the control and CCl4-intoxicated groups. UCSCs: Umbilical cord stem cells, HLCs: Hepatocyte-like cells

Normal liver architecture was observed in control group. Lobular structures of the liver were damaged in CCl4-intoxicated rats. Accumulation of erythrocytes, infiltration of inflammatory cells, nuclear pyknosis and sinusoidal dilation were observed in this group. In cell-free scaffold transplanted animals, histological criteria were similar to those in the CCl4-intoxicated rats. The histological criteria significantly improved in both UCSCs and HLCs transplanted-animals in comparison with the CCl4-treated rats. The liver histology was the same in the UCSCs and HLCs transplanted-rats (Figure 5 and Table 1).

Figure 5

Light microscopy of cross sections of H&E stained liver tissue. A: CCl4 group; B: cell-free beads transplanted group; C: Umbilical cord stem cells (UCSCs) beads transplanted group; D: Hepatocyte-like cells (HLCs) beads transplanted group. C: congestion of RBCs, I: infiltration of inflammatory cells, P: nuclear pyknosis, S: sinusoidal dilation

Table 1

Histological criteria in control and experimental groups

Histological criteria	Groups
	Control	CCl4	Cell-free beads	UCSCs beads	HLCs beads
Congestion of RBC	0.0 ± 0.0	2.5 ± 0.3*	2.8 ± 0.12*	0.41 ± 0.4*##	0.35 ± 0.04*##
Infiltration of inflammatory cells	0.01 ± 0.001	1.8 ± 4.16*	2.1 ± 4.3*	0.43 ± 0.05*##	0.2 ± 0.09*##
Sinusoidal dilatation	0.04 ± 0.002	2.48 ± 0.25*	2.53 ± 0.19*	1.4 ± 0.05*#	1.35 ± 0.07*#
Pyknosis (%)	0.01 ± 0.002	36.3 ± 5.3*	38.3 ± 3.8	13.9 ± 2.6*##	12.3 ± 2.9*##

Values are expressed as mean ± SD.

* and # symbols respectively indicate comparison to the control and CCl4-intoxicated rats groups. UCSCs: Umbilical cord stem cells, HLCs: Hepatocyte-like cells.

P < 0.001,

P < 0.05,

P < 0.01;

In cell-free scaffold transplanted group, expression of ALB, AFP and CK-18 was similar to the CCl4-treated animals. In both UCSC and HLC transplanted-groups, ALB and CK-18 expression significantly increased, while AFP expression significantly decreased compared to the CCl4-intoxicated rats. There was no significant differentiation in expression of AFP between UCSCs and HLCs transplanted groups. Expression of ALB and CK-18 genes in HLCs transplanted-group significantly increased compared to the UCSCs-transplanted animals (Figure 6).

Figure 6

Gene expression in treated groups. Values expressed as mean±SD for 6 rats. *P<0.05, **P<0.01, ***P<0.001, # P<0.05; * and # symbols respectively indicate comparison to the control and CCl4-intoxicated groups. UCSCs: Umbilical cord stem cells, HLCs: Hepatocyte-like cells

Discussion

The results of this study demonstrated that administration of both UCSCs and HLCs improve the liver functions. Hu et al. (2015) have reported that somatic stem cells could effectively differentiate in alginate micro beads into HLCs and secret ALB (23). The HLCs encapsulated in alginate scaffolds alleviated ALF induced by CCl4 when transplanted into the liver of rats. In a study, injection of UCSCs-derived HLCs effectively improved CCl4–induced liver damage in mice (24). Transplantation of the encapsulated hepatocytes in alginate/poly-L-lysine improved the hepatic function in a 90% partial hepatectomy of rats (14). Therefore, cell therapy could ameliorate CCL4-incuced hepatotoxicity.

In this study, ALT and AST levels that are important indicators of liver injury were decreased in UCSC and HLC-treated animals. The serum activity of these enzymes is routinely measured for the detection of liver disorders (25). The liver cells release these enzymes into the blood stream following injury or death. ALP levels also decreased in treated animals. In acute hepatitis, presenting cholestasis and peripheral arterial disease, ALP usually remains normal or moderately increased (25). Reversal of the elevation of plasma enzyme activity in CCl4-induced hepatotoxicity indicates that the transplanted cells may decrease liver cell death.

AFP significantly decreased in both UCSCs and HLCs transplanted-rats. In addition, ALB secretion significantly increased after transplantation of these cells. This indicates that both UCSCs and HLCs have hepato-protective effects.

The results of the histological study also support the results of biochemical parameters. Transplantation of HLCs effectively attenuated histological changes such as nuclear pyknosis, sinusoidal dilation, congestion of RBCs, and infiltration of inflammatory cells compared to the rats treated with CCl4 alone.

The alteration in ALB secretion and gene expression was accompanied by histological improvement in both UCSC and HCLs transplantation. Transplantation of hepatic-differentiated human amniotic epithelial cells in a mice cirrhosis model led to a successful in vivo engraftment and differentiation into functional hepatocytes (26).

The present study showed that the encapsulated UCSCs also attenuated ALF when transplanted into the liver. Li et al. (2014) showed that hydrogel scaffolds loaded with UCSCs significantly improved liver damage (8). It has been shown that intraportal transplantation of human bone marrow MSCs prevents hepatocellular degeneration induced by D-galactosamine in pigs (27). Findings have also shown that fluorescently labeled MSCs can accumulate in liver and alleviate CCl4-induced liver damage in mice (28). In agreement with our findings, Zhou et al. (2014) have demonstrated that UCSCs and UCSCs-derived hepatocytes have similar hepato-protective effect in an ALF mice model (29).

Although both UCSC and HLC transplantation ameliorated the adverse impact of CCl4, the biochemical tests revealed a significant reduction of ALB secretion in UCSC-treated animals compared to the HLCs-transplanted animals.

Transplantation of human induced pluripotent stem cells -derived HLCs attenuated ALT, AST and BUN levels in ALF-induced D-galactose-treated rats. These cells also markedly increased ALB secretion in D-galactose-treated rats (30) and CCl4 treated-mouse (31). Injection of UCSCs and adipose stem cells via the tail vein improved liver failure, and decreased ALT and AST levels in CCl4-treated mice (32, 33).

In the present study, UCSCs in alginate beads expressed hepatocyte-specific markers (CK-18 and ALB) after two weeks of transplantation in CCl4-treated rats. However, we could not clearly prove that the transplanted UCSCs were fully differentiated into cells with functions comparable to endogenous hepatocytes.

In the current study, it was found that transplantation of UCSCs markedly improved liver functions and also increased ALB, AFP and CK18 expression level. This finding indicates that the UCSCs may differentiate into the HLCs in liver niche or produce cytokines that act as chemoattractant for hepatocyte progenitor cells. It is also possible that stimulating factors such as complement system, platelets, inflammatory cytokines, growth and anti-inflammatory factors (34) that are secreted from damaged liver tissue affect UCSCs and induce differentiation of these cells into HLCs. Besides, it has been reported that 3D culture system influences the liver-specific marker expression pattern in the absence or presence of chemical induction (35). Therefore, 3D culturing may also promote the differentiation toward hepatocytes. However, our methodology did not support these declarations.

We showed the anti-inflammatory effects of both HLCs and UCSCs on ALF liver according to scoring system. There are some reports that also confirm the anti-inflammatory effects of both USCSs and HLCs after transplantation. (36). Tuo et al. (2017) showed that UCSCs can inhibit immune response, promote hepatocyte regeneration, alleviate the progression of liver fibrosis, and improve liver function in CCl4-induced hepatotoxicity (37).

Tumor formation is the most important problem of cell therapy. Tumorigenicity is a common problem of embryonic stem cells or induced pleuripotent stem cells transplantations. However, tumorigenicity of MSCs is very low (38). It was previously reported that differentiated MSCs were more stable and safer than undifferentiated MSCs (7).

There are many transplantation sites, including omentum, small intestinal mesentery, and subcutaneous space of the abdominal wall (39). In most studies, the intraperitoneal cavity is used as the site of transplantation, which may cause fibrosis around the encapsulated cells (14). Transplantation of the adipose-derived stem cells in the spleen, as another transplantation site, has been reported to fail the delivery of the cell to the liver (40). This study suggests that the insertion of cells in the alginate scaffolds into the liver is better than other sites.

Conclusion

This study demonstrated that transplantation of UCSCs and HLCs in alginate scaffolds improve CCl4-induced ALF. It seems that USSCs is as good as HLCs in curing ALF. However, the mechanisms such as paracrine, anti-inflammatory, and antiapoptotic effects of transplanted UCSCs and HLCs in alginate beads remain to be resolved.