How important is differentiation in the therapeutic effect of mesenchymal stromal cells in liver disease?

Cytotherapy, 2013; 0: 1e10

How important is differentiation in the therapeutic effect of mesenchymal stromal cells in liver disease?

HANYU WANG1, TINGTING ZHAO1, FANG XU1, YAN LI1, MINGYUAN WU1,2,3, DELIN ZHU1, XIULI CONG1,4 & YONGJUN LIU1 1

Alliancells Institute of Stem Cells and Translational Regenerative Medicine, Tianjin, China, 2Beijing Alliancells-PuRui Bioscience Co, Ltd, Beijing, China, 3University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA, and 4University of Florida, Department of Medicine, Gainesville, Florida, USA

Abstract Background aims. The protocols for differentiation of hepatocyte-like cells (HLCs) from mesenchymal stromal cells (MSCs) have been well established. Previous data have shown that MSCs and their derived HLCs were able to engraft injured liver and alleviate injuries induced by carbon tetrachloride. The goal of the current study was to determine the differences of MSCs and their derived HLCs in terms of therapeutic functions in liver diseases. Methods. After hepatic differentiation of umbilical cordederived MSCs in vitro, we detected both MSC and HLC expressions of adhesion molecules and chemokine receptor CXCR4 by flow cytometry; immunosuppressive potential and hepatocyte growth factor expression were determined by means of enzyme-linked immunosorbent assay. We compared the therapeutic effect for fulminant hepatic failure in a mouse model. Results. MSC-derived-HLCs expressed lower levels of hepatocyte growth factor, accompanied by impaired immunosuppression in comparison with MSCs. Furthermore, undifferentiated MSCs showed rescuing potentials superior to those in HLCs for the treatment of fulminant hepatic failure. Conclusions. After differentiation, HLCs lost several major properties in comparison with undifferentiated MSCs, which are beneficial for their application in liver diseases. Undifferentiated MSCs may be more appropriate than are HLCs for the treatment of liver diseases. Key Words: cytotherapy, differentiation, liver diseases, mesenchymal stromal cells

Introduction Studies have shown that human embryonic stem cells (ESCs) (1,2) and adult stem cells (3,4) in culture could be differentiated into functional hepatocytelike cells. It has been reported that in partial hepatectomy combined with monocrotaline pretreatment in an immunodeficient Pfp/Rag2e/e mouse model, 10e20% of hepatocytes in the host liver are replaced by hepatocyte-like cells (HLCs) differentiated from mesenchymal stromal cells (MSCs) after 10 weeks (5). These results indicate that HLCs derived from stem cells may be a new, suitable candidate for liver diseases (4), especially fulminant hepatic failure (FHF) and end-stage liver diseases (6). Generated populations can be easily contaminated by undifferentiated ESCs, which have the capacity to induce the formation of teratomas (7,8). Adult stem cells are explored as a promising source of cells in the clinical research without ethic problems and with the potential of neoplastic transformation of

human ESCs (9). MSCs not only have capability to engraft, repopulate damaged tissue and restore function in animal models (10e12) but also could reduce resident cells death and facilitate endogenous regeneration (13,14). Additionally, MSCs could be easily expanded in vitro. These special properties of MSCs make them be the best source of cell therapy in various diseases, including liver cirrhosis and liver failure. The differentiation protocol of HLCs from MSCs has been well documented (3). Differentiated HLCs were able to engraft the injured liver and secreted albumin (ALB) in vivo (5,10,15). HLCs from human adipose tissueederived MSCs alleviated acute liver injury induced by carbon tetrachloride in nude mice (16). Both MSCs and HLCs have been proven to have therapeutic effects for liver diseases, such as liver fibrosis (17e19), liver injury (20,21), FHF (10,16,22) and liver regeneration (5,23,24). The goal of the current study is to compare therapeutic effects on liver diseases

Correspondence: YongJun Liu, PhD, Alliancells Institute of Stem Cells and Translational Regenerative Medicine, 45 Dong Jiu Dao, Airport Economic Park, Tianjin, 300381, China. E-mail: [email protected] (Received 18 March 2013; accepted 29 July 2013) ISSN 1465-3249 Copyright Ó 2013, International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jcyt.2013.07.011

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Figure 1. UC-MSCs could be induced to differentiate into functional HLCs. After hepatic differentiation, UC-MSCs changed from fibroblast-like to larger, round, epithelial-like morphology. (A) Undifferentiated UC-MSCs; (B) HLCs; (C) ALB expression detected by immunofluorescence microscopy; (D) CK-19 expression detected by immunofluorescence microscopy; (E) undifferentiated UC-MSCs were not able to synthetize glycogen; (F) HLCs were strongly positive for glycogen; (G) urea concentration was measured in supernatant during the process of hepatic differentiation at the points of 6 days, 10 days, 24 days, 40 days and 50 days. Differentiated cells could synthesize urea in a time-dependent manner; (H) HLCs showed characteristic capacity of low-density lipoprotein uptake; (I) both MSCs and HLCs expressed ALB detected by flow cytometric analysis.

between MSCs and HLCs. We determined that MSCs are more effective than are HLCs in the treatment of FHF induced by D-galactosamine (DGalN) and lipopolysaccharide (LPS). The immunosuppressive capacity of MSCs was impaired after hepatic differentiation. Therefore, undifferentiated MSCs may be more appropriate than are HLCs in the treatment of liver diseases.

Methods Umbilical cordederived MSC preparation Umbilical cords were collected from healthy full-term pregnant donors with written consent and approval by the institutional review board of the Alliancells Institute of Stem Cells and Translational Regenerative Medicine. Umbilical cordederived MSCs (UC-MSCs) were cultured in complete culture medium containing Dulbecco’s minimum essential

medium/F12 (1:1) (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (HyClone, Logan, UT, USA), 100 U/mL penicillin-streptomycin (Sigma Aldrich, St Louis, MO, USA), 1% glutamine (Sigma Aldrich) and 10 ng/mL epidermal growth factor (Sigma Aldrich). UC-MSCs were isolated and expanded and characterized as described previously (25,26). UC-MSCs at passages 3e6 were used for experiments unless otherwise stated. Hepatic differentiation of UC-MSCs in vitro Hepatic differentiation of human UC-MSCs was performed by use of a slightly modified two-step protocol (3). Briefly, UC-MSCs were first cultured in D/F-12 containing 50 ng/mL HGF (Peprotech, Rocky Hill, NJ, USA), 10 ng/mL basic fibroblast growth factor, 10
8 mol/L dexamethasone (Sigma Aldrich) and 50 mg/mL insulin-transferrin-selenium premix (Gibco) for 2 weeks. The cells were then

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Figure 2. Both undifferentiated UC-MSCs and HLCs have the potential of multipotent differentiation and could differentiate into osteoblasts and adipocytes. Surface characteristic markers of two types of cells were detected by flow cytometric analysis. Except for CD90, CD73 and CD105 expressions were dramatically decreased after differentiation of UC-MSCs. HLA-DR expression was increased. This experiment was representative of three performed. (A) Alizarin red S staining in UC-MSCeundifferentiated UC-MSCs; (B) oil red O staining in UC-MSC HLCs. (C) Alizarin red S staining in HLCs; (D) oil red O staining in HLCs; (E) surface characteristic markers of UCMSCs; (F) surface characteristic markers of HLCs.

cultured in the maturation medium that contained 20 ng/mL oncostatin M (R&D, Minneapolis, MN, USA), 10
6 mol/L dexamethasone and insulintransferrin-selenium for another 4 weeks. Medium changes were performed twice weekly. Induction of HLCs was assessed by in vitro assays for characteristic functions of hepatocytes, such as ALB expression, urea secretion, uptake of low-density lipoprotein and polyglycans synthesis.

Flow cytometry Cells were harvested and stained with fluorochrome-conjugated antibodies and analyzed with FACSCalibur (BD Biosciences, San Jose, CA, USA). Antibodies specific for ALB and the adhesion molecules CD29, CD44, CD49d, CD54 CD56, CD90, CD105, CD106 and CD166, and for MSCs characterization, CD11b, CD19, CD34,

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Figure 3. Cytokines in supernatants were detected by ELISA. Differentiation impaired the immunosuppression power of MSCs on CD4þ T-cells. After differentiation, UC-MSCs decreased the powerful immunosuppressive capacity. Values shown represent mean  standard error of the mean, n ¼ 6 wells. *P < 0.05; **P < 0.01.

CD45, CD73, CD90, CD105 and HLA-DR, were used. All of the direct fluorochrome-conjugated antibodies were purchased from BD Company. Cells and CD4þ T cell co-culture experiment First, peripheral blood mononuclear cells were isolated by Ficoll-Paque (GE Healthcare, Piscataway, NJ, USA) density gradient centrifugation with blood donated from health volunteers. CD4þ T cells were highly purified from peripheral blood mononuclear cells by use of the CD4þ T-cell isolation kit II (negative selection) according to the manufacturer’s instruction (Miltenyi Biotec, Bergisch Gladbach, Germany). Irradiated (20 Gy) cells (UC-MSCs and HLCs) (1  104 cells/well) co-incubated with CD4þ T cells (1  105cells/well) in Roswell Park Memorial Institute 1640 medium (Gibco) containing 10% fetal bovine serum for 3 days in 96-well plates (Corning, Corning, NY, USA). Phytohemagglutinin A (10 mg/mL) was used to stimulate activation of CD4þ T cells. Culture supernatants were collected, and the concentrations of interferon (IFN)-g were measured by means of enzyme-linked immunoassay (ELISA) (R&D). HGF expression: ELISA assay On the basis of its vital role in liver tissue, HGF expression was evaluated; 5  105 UC-MSCs (P3)

Figure 4. HGF expression of three types of cells in supernatant was detected by ELISA. HGF expression of differentiated cells dramatically decreased, compared with that in undifferentiated UC-MSCs. Values shown represent mean  standard error of the mean, n ¼ 6 wells. **P < 0.01.

were seeded in 25-cm2 flasks (Corning) in 5 mL of fresh D/F-12 medium for 3 days before initiation of differentiation. When UC-MSCs differentiated into HLCs, the passages of cells reached approximately 15. At the end of differentiation, 5  105 cells (UC-MSCs, P15 and HLCs) were seeded in 25-cm2 flasks (Corning) in 5 mL of fresh D/F-12 medium for 3 days. HGF secretion in the supernatant was measured by use of an ELISA kit (Invitrogen, Eugene, OR, USA). Measurement for each group of different cells was repeated five times (n ¼ 6). FHF model Balb/c male mice were given intraperitoneally administrated D-galactosamine (D-GalN, 800 mg/ kg) and LPS (50 mg/kg), each dissolved in D-Hanks’ solution. Animal experiments were performed under approved protocols for the humane treatment of animals, regulated by the Alliancells Institute. Ten-week-old mice were separated randomly into three groups. The control group had 20 mice, and each cytotherapy group had 10 mice. These mice were observed every 24 h for 4 days after the D-GalNþLPS injections. Mice were treated 1 h after FHF induction with intravenous infusion of

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Figure 5. To evaluate cell migration ability, several adhesion molecules and CXCR4 were detected by flow cytometric analysis. In vivo migration assay was performed. The migration ability of HLCs was dramatically reduced, compared with undifferentiated UC-MSCs. (A) Expressions of several adhesion molecules from undifferentiated UC-MSCs and (B) from HLCs; (C) CXCR4 expression in UC-MSCs and HLCs; (D) MSCs labeled with CM-DiI engrafted into injured liver; (E) HLCs labeled with CM-DiI engrafted into injured liver; percentages of D and E are derived from the rate of red/blue.

cells (UC-MSCs and HLCs) at a dose of 1  106 and 1  105 cells, respectively. For evaluating serum indexes of liver function, we detected the concentrations of aspartate aminotransferase (AST) and alanine transaminase (ALT) in blood serum at the point of 8 h after Gal-N/LPS injection. Frozen sections of liver tissues were used to perform hematoxylin and eosin staining and cell death immunohistochemical staining according to an in situ cell death detection kit (TUNEL Technology; Roche, Mannheim, Germany). Brown staining of DNA represented dead cells under light microscopy.

Chemotaxis assay in vivo To test the migration ability of undifferentiated UC-MSCs and HLCs, cells were labeled with CellTracker chloromethylbenzamido (CM-DiI) (Invitrogen), which was incorporated into cell membranes and detected by fluorescence microscopy. Briefly, 5 mL of CM-DiI labeling solution per milliliter of medium was added into cell culture medium. Subsequently, cells were incubated for 5 min or less at 37 C and then for an additional 15 min at 4 C. After labeling, cells were washed with phosphate-buffered saline and collected by means of trypsin digestion.

CM-DiIelabeled cells (2  106 UC-MSCs and HLCs) were transplanted into FHF mice 1 h after induction. After 24 h, mice were euthanized to perform liver frozen-section examination after hepatic perfusion with phosphate-buffered saline. Quantitative percentage of cell numbers was performed by use of the public software ImageJ. Statistical analysis Results are expressed as mean  standard error of the mean. Statistical comparison of the data was performed with the use of one-way analysis of variance for comparison of more than two groups. Differences were considered significant for values of P < 0.05. Results Hepatic differentiation of UC-MSCs in vitro UC-MSCs derived from the human umbilical cords showed positive expression (>95%) for the markers of CD73, CD90 and CD105 but negative (<2%) for the markers of CD34, CD45, CD11b, CD19 and HLA-DR, consistent with the definition by the International Society for Cellular Therapy (27). When cultured under standard in vitro differentiating

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Figure 6. According to liver histochemical staining and survival rate, MSCs showed highly effective therapy for FHF, compared with HLCs. (A) Hematoxylin and eosin (HE) staining, normal liver; (B) HE staining, FHF model liver; (C) HE staining, UC-MSCetreated liver; (D) HE staining, HLC-treated liver; (E) in situ cell death detection, normal liver; (F) in situ cell death detection, FHF model liver; (G) in situ cell death detection, UC-MSCetreated liver; (H) in situ cell death detection, HLC-treated liver; (I) biochemical analysis of ALT and AST in blood serum. (J) High dose of cells (1  106) for FHF treatment; (K) low dose of cells (1  105) for FHF treatment.

conditions, MSCs were able to differentiate into osteoblasts, adipocytes and chondroblasts, respectively, as described previously (25,26). In the present study, we used UC-MSCs to perform hepatic differentiation as previously described (3). During the process of hepatic differentiation, UC-MSCs changed in morphology from fibroblast-like to larger, round, epithelial-like cells (Figure 1A,B). After 6 weeks in hepatic differentiation culture, the cells showed strong expressions of ALB and CK19 by immunofluorescence microscopy (Figure 1C,D). To test hepatocellular metabolic functions, the cells were stained for polyglycans, an indicator of glycogen deposition. Undifferentiated UC-MSCs were not able to synthetize glycogen (Figure 1E); on the contrast, the differentiated cells were strongly positive for glycogen (Figure 1F). The differentiated cells also synthesized urea in a time-dependent manner, which further

confirmed the hepatocellular metabolic functions of differentiated cells (Figure 1G). Furthermore, when the cells were cultured in the presence of dil complex acetylated low density lipoprotein (Dil-Ac-LDL), they showed characteristic capacity of low-density lipoprotein uptake (Figure 1H). However, undifferentiated UC-MSCs also express high levels of ALB (Figure 1I), which indicates that ALB may not be suitable as a characteristic indicator of hepatic differentiation. These results demonstrated that UCMSCs could differentiate into hepatocyte-like cells with distinct hepatocytic characteristics. When UC-MSCs underwent hepatic differentiation, change of the surface expressions of characteristic markers was observed (Figure 2A,B). The expression levels of CD90, CD73 and CD105 expressions on HLCs were dramatically decreased, along with the increased HLA-DR level, which indicates that the immunogenicity of HLCs was higher

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Figure 6. (continued).

than that of UC-MSCs. However, HLCs still have the potential of multipotent differentiation, which was demonstrated by their differentiation into osteoblasts and adipocytes (Figure 2CeF). Suppression on CD4þ T-cell activation Numerous reports and our previous data have certified that UC-MSCs strongly suppress the activation of T cells by soluble factors (28e31). The immunosuppressive capacity was investigated by coculturing cells with CD4þ T cells. When CD4þ T cells were stimulated with the mitogen phytohemagglutinin A, they were activated and produced large amounts of IFN-g. The level of IFN-g represented the activation state of CD4þ T cells (32). To compare the functional difference between MSCs and HLCs, we conducted the IFN-g secretion assay

to evaluate their immunosuppressive capacity. CD4þ T cells secreted high levels of IFN-g when cultured alone, but the production was significantly suppressed in the co-culture with UC-MSCs (Figure 3); in contrast, the secretion of IFN-g was still present in a substantial amount when cocultured with HLCs (Figure 3). These data indicate that HLCs partially lose the powerful immunosuppressive capacity of MSCs. HGF expression HGF is the most important agent to promote the proliferation of hepatocytes (33,34) and liver regeneration (33,35). Our previous data have shown that UC-MSCs secreted high amounts of HGF (30). We were intrigued to find that the amount of HGF secreted from UC-MSCs was higher than that from

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bone marrowederived MSCs (36). In the present study, we found that the expression level of HGF was dramatically decreased when UC-MSCs differentiated into HLCs (Figure 4), which suggests that UCMSCs are more appropriate than are HLCs to facilitate hepatocyte proliferation. Migration ability MSCs have the capability to engraft, repopulate the damaged tissue and restore function in animal models (10,11,37), and their migration ability is closely related to that in adhesion molecules and receptors of chemokine. Undifferentiated UC-MSCs expressed numerous adhesion molecules, such as high levels of CD29, CD44, CD49d, CD54, CD56, CD90, CD105 and CD166, but a low level of CD106 (Figure 5A). However, the expressions of these adhesion molecules in HLCs were dramatically decreased, compared with UC-MSCs (Figure 5B). Consistent with the reduction of adhesion molecules, the expression level of chemokine receptor CXCR4 (CD184) was also decreased in HLCs (Figure 5C), which indicates that the migration ability of HLCs was reduced. To further evaluate in vivo migration ability, UCMSCs and HLCs cells were labeled with CellTracker CM-DiI and injected into FHF mice with severe liver injury. As expected, both UC-MSCs and HLCs could migrate into the injured liver; however, UCMSCs (Figure 5E) showed more migration ability than did HLCs (Figure 5D) in injured liver sections. Rescuing potentials It was reported that recipient survival was apparently enhanced by intravenous injection compared with intrasplenic infusions (10). Thus, after induction of FHF, cells (both UC-MSCs and HLCs) were injected through the mouse tail vein. The high mortality rate of FHF resulted from the rapid development of hepatocellular dysfunction or severe necrosis of hepatocytes. Both UC-MSCs and HLCs could rescue hepatocytes apoptosis/necrosis (Figure 6). After D-GalN/LPS injection, the concentrations of ALT and AST in blood serum dramatically increased (Figure 6I). However, both MSC (1  106) and HLC (1  106) treatments were equally able to reduce ALT/AST concentrations, which suggests that these two types of cells effectively prevent hepatocytes from injury. After two doses of cells, UC-MSCs showed higher effective therapy for FHF, compared with that in HLCs (Figure 6J,K). The survival rate of FHF reached to 100% at the dose of 1  106 MSCs, whereas in the HLC group, it reached only 70% (Figure 6J). At the low dose of 1  105 cells, the UC-MSCs group achieved 60% survival, whereas 40% survival was

reached in the HLC group (Figure 6K). These data suggest that undifferentiated UC-MSCs are more effective than are HLCs in the treatment of FHF. Discussion MSCs have recently shown a promising prospect in cell therapy because they have the powerful functions of immunoregulation (28,29) and tissue repair (13,38e40). Accumulating data have shown that MSCs ameliorated liver function without cellular fusion in different animal models (5,10,15). Therefore, the possible mechanisms may be due to the secreted soluble factors that enhance repair of the damaged tissues resulting from suppressing inflammatory and immune reactions or by stimulating propagation and differentiation of tissue-endogenous stem cells (41,42). This hypothesis was also supported by animal model experiments (22,24). On the basis of this hypothesis, we must look at the changes of secreted soluble factors before specific differentiation is performed. In the matter of liver diseases, we must evaluate the change of HGF expression. It is widely accepted that HGF is the most important agent promoting the proliferation of hepatocytes (33,34) and liver regeneration (33,35). HGF stimulates proliferation of hepatic progenitor cells to repair hepatic injury (43). HGF also protects against Fas-mediated hepatocytes apoptosis (44), leading to rescue from Fas-induced FHF (45). Thus, HGF plays an important role in the mechanism(s) of cytotherapy for liver diseases. We observed that UC-MSCs express high levels of HGF, in accord with a previous study (36). Furthermore, the expression of HGF by UC-MSCs is 30 times higher than that of bone marrow derivedeMSCs (36). Unfortunately, when MSCs differentiated into HLCs, HGF expression was decreased dramatically (Figure 4). Previous data have shown that approximately 80% of the cells were ALB-positive after hepatic differentiation (46). However, we found that >98% of undifferentiated UC-MSCs expressed ALB and were enhanced by hepatic differentiation (Figure 2G). Although HLCs obtained the ability to synthetize glycogen, HLCs lost most migration ability, which contributes to enhanced repair of the damaged tissues. When UC-MSCs differentiated into HLCs, adhesion molecules (Figure 5A,B) and chemokine receptor CXCR4 (Figure 5C) expression was decreased. Approximately 8.2% of the total cells in liver were derived from HLCs (Figure 5D), compared with 22.7% of undifferentiated UC-MSCs (Figure 5E). Furthermore, undifferentiated UC-MSCs showed rescuing potentials superior to those of HLCs in the treatment of FHF (Figure 6). Another previous

Differentiation in therapeutic effect of MSCs in liver disease in vivo study also demonstrated that MSCs achieved better rescuing efficacy compared with that of HLCs derived from MSCs (10). Additionally, MSC transplantation enhanced the repopulation of endogenous liver cells. In comparison, HLC-recipient liver showed few signs of regeneration of the necrotized regions (10). We were intrigued to find that undifferentiated UC-MSCs also expressed ALB, which is beneficial for liver fibrosis and cirrhosis (Figure 1I). In summary, UC-MSCs lost several important characteristic properties when differentiating into HLCs. For example, HGF expression was decreased dramatically, accompanied by the impairment of immunosuppression; migration ability and the efficacy of treatment for FHF were decreased. Thus, our data provide further evidence that undifferentiated UC-MSCs may be more appropriate than HLCs in terms of the treatment of liver diseases. Acknowledgment This study was supported by a grant from National Natural Science Foundation of China (Grant No. 30872618). Disclosure of interests: The authors have no commercial, proprietary, or financial interest in the products or companies described in this article.

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