Mol Cell Biochem DOI 10.1007/s11010-014-2147-7

Up-regulation of CXCR4 in rat umbilical mesenchymal stem cells induced by serum from rat with acute liver failure promotes stem cells migration to injured liver tissue Changqing Deng • Ailan Qin • Weifeng Zhao Tingting Feng • Cuicui Shi • Tao Liu

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Received: 12 March 2014 / Accepted: 11 July 2014 Ó Springer Science+Business Media New York 2014

Abstract The role of C-X-C chemokine receptor type 4 (CXCR4) in umbilical mesenchymal stem cells (UMSCs) as therapy for liver disease is ill understood. The aim of the study was to evaluate rat UMSCs (rUMSCs) on CXCR4 expression and homing to injured liver tissue. rUMSCs were isolated from umbilical cords of pregnant rats. Acute liver failure (ALF) models were developed using D-galactosamine. CXCR4 expression induction by serum from rats with ALF (LFS), cytokines, growth factors, and LPS was analyzed. CXCR4 expression was analyzed by RT-PCR, western blot, and flow cytometry. rUMSCs were labeled with carboxyfluorescein and pretreated with LFS to induce CXCR4 expression and were transplanted into ALF rats. Animals were sacrificed 48 h and 1 week after transplantation. Liver-homing rUMSCs were observed under fluorescence microscopy. rUMSCs were successfully isolated, expressing CD90 and CD106, but not CD34 and CD45. mRNA and protein expressions of CXCR4 were strongly up-regulated by LFS and by the mixture of cytokines, stem cell factor, and LPS (CM). Expression of cell surface CXCR4 on rUMSCs in groups treated with LFS (42.37 ± 1.60 %) and CM (40.17 ± 1.78 %) was higher than that in the untreated control group (9.67 ± 1.06 %) (both P \ 0.001). At 48 h after Electronic supplementary material The online version of this article (doi:10.1007/s11010-014-2147-7) contains supplementary material, which is available to authorized users. C. Deng  A. Qin (&)  W. Zhao  T. Feng  C. Shi  T. Liu Department of Infectious Diseases, First Hospital Affiliated to Suzhou University, Suzhou 215006, Jiangsu, China e-mail: [email protected] C. Deng Department of Gastroenterology, Affiliated Hospital of Jiangxi University of Traditional Chinese Medicine, Nanchang 330020, Jiangxi, China

transplantation, more rUMSCs pretreated with LFS appeared in the portal area, and migrated to the liver parenchyma after 1 week. LFS strongly induced the surface expression of CXCR4 on rUMSCs. Increasing CXCR4 expression on rUMSCs may enhance their homing ability to injured liver tissue, and may eventually be used for treating liver diseases. Keywords Umbilical cord  Mesenchymal stem cell  CXCR4  Homing  Acute liver failure

Introduction Liver failure is one of the leading causes of death worldwide [1]. Orthotopic liver transplantation is an effective treatment for liver failure, but the shortage of donor livers is the major obstacle to this procedure. Liver cell transplantation is also restricted due to the poor availability of donor organs for the isolation of primary human hepatocytes [2]. In addition, on average, only 30 % of hepatocytes survive transplantation. Therefore, successful engraftment often depends on the number of infused hepatocytes [3]. Stem cells are an alternative source of hepatocytes for liver cell therapies, and are currently being investigated to overcome the donor organ shortage. Mesenchymal stem cells (MSCs) originating from mesoderm are excellent candidates for cell therapy due to their low immunogenicity. MSCs have the capacity to differentiate into hepatocyte-like cells [4–6]. Umbilical MSCs (UMSCs) derived from Wharton’s jelly of umbilical cord can also be induced into hepatocyte-like cells using hepatocyte growth factor (HGF), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and oncostatin M (OSM) [5, 7–9]. The main advantages of using UMSCs are that they are free from ethical concerns and can be easily obtained.

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However, the homing mechanisms of MSCs are still unclear. The stromal cell-derived factor-1 (SDF-1)/G-protein-coupled C-X-C chemokine receptor type 4 (CXCR4) axis plays an important role in MSCs homing to injured tissues [10, 11]. The interaction between SDF-1 and CXCR4 mediates trafficking of rat MSCs to the sites of injury, and the migration of MSCs in vitro. Homing capabilities of MSCs are greatly influenced by cell culture conditions. Culturing MSCs for more than two passages were associated with a decreased adhesion molecules expression, and loss of chemokine receptors, including CXCR4 [11, 12]. The loss of these chemokine receptors results in homing impairment and represents a substantial challenge for the therapeutic application of MSCs. Increased CXCR4 expression in UMSCs and leukocytes improve their homing efficiency [13–18]. Kyriakou et al. [19] studied a CXCR4 overexpression system in mesenchymal stem cell to demonstrate CXCR4 role in promoting homing. Currently, xenogeneic transplantation study using human MSCs injected in mice models of hepatic diseases had the disadvantage of inter-species differences, influencing homing efficiency, immunological regulation, and therapeutic efficacy [4, 14, 20, 21]. Results from studies using immunodeficient rats or nude mice as recipient cannot be directly transposed to humans since patients with liver failure or cirrhosis are not immunodeficient and because immunodepressors cannot be administered when they are transplanted with UMSCs. In order to emulate a clinical context, allogeneic UMSCs should be used for transplantation in animal models. Many studies assessed rat bone marrow MSCs for tissue repair, but few assessed rat UMSCs (rUMSCs) [22]. Indeed, Ganta et al. [22] reported that rUMSCs completely abolished rat mammary carcinomas. Wang et al. [23] showed that rUMSCs could be used to treat pancreas injury. Therefore, the aim of the present study was to evaluate CXCR4 expression in rUMSCs, and their homing to injured liver tissue. The present study was a preclinical one that was aiming toward clinical applications. Since an overexpression system cannot be used to promote CXCR4 expression in a clinical setting, we used naturally occurring CXCR4 expression, and serum from ALF rats was used as the stimulating agent. In clinical practice, we could use the patient’s own serum to induce CXCR4 expression. Results from the present study could eventually lead to new acute liver failure (ALF) treatments to overcome donor shortage.

Materials and methods Animals Fifty male Sprague–Dawley (SD) rats (weighing 198.4 ± 8.5 g) and ten ED 25 pregnant SD rats were

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supplied by the Shanghai Si Ke Lai Si Medical Experimental Animal Center (Shanghai, China) (SCXK 2007-0005), and were housed at 20–25 °C, 50 ± 5 % humidity, 12:12 h light/dark cycle, and with ad libitum access to food and water. All animal experiments complied with ethical requirements, and were approved by the ethics committee of the Suzhou University (Suzhou, China). Isolation and culture of rUMSCs rUMSCs were obtained as previously published using a twostep digestion [24]. Umbilical cords (2.0–2.5 cm) of pregnant SD rats were collected in 0.9 % sterile NaCl solution, and the blood was washed out. Cords were cut into 1-mm3 pieces, immediately transferred to a 15 ml centrifuge tube and centrifuged at 2509g for 5 min at room temperature. The pellet was suspended in D-Hank’s balanced salt solution supplemented with 1 mg/ml of type IV collagenase (GIBCO, Invitrogen Inc., Carlsbad, CA, USA). Tissues were digested at 37 °C in a 5 % CO2 atmosphere for 1 h. The supernatant was collected and placed at 4 °C. The pellet was added with 0.25 % trypsin-0.02 % EDTA in D-Hank’s solution and digested at 37 °C in 5 % CO2 for 30 min. Digestion was stopped with DMEM-Low glucose (LG) (GIBCO, Invitrogen Inc., Carlsbad, CA, USA) supplemented with 10 % FBS (GIBCO). Undigested tissue was filtered using 200 lm filters. The homogenate and previously reserved supernatant were mixed and centrifuged at 1,2009g for 5 min. The cell pellet was resuspended in a growth medium composed of DMEM-LG with 2 mmol/l L-glutamine supplemented with 10 % FBS, 100 U/ml of penicillin, and 100 lg/ml of streptomycin (Invitrogen Inc., Carlsbad, CA, USA). After cell counts and concentration adjustments, cells were seeded in 25 cm2 culture flasks at a density of 5 9 105 cells/cm2, and cultured in expansion medium at 37 °C in a humidified 5 % CO2 atmosphere. Culture medium was replaced after 3 days, and then every 4 days afterward, or when the color of the medium changed to yellow. At 80–90 % confluence, cells were detached from the flask using a 0.25 % trypsin-0.02 % EDTA solution. Cells were placed at a density of 1 9 104 cells/cm2 in growth medium, and medium was changed twice a week. Identification of rUMSCs After three passages, rUMSCs (1 9 106 cells/ml) were stained with phycoerythrin (PE)-conjugated monoclonal antibody against CD106 or CD90 (Biolegend, San Diego, CA, USA), or with fluorescein-isothiocyanate (FITC)conjugated monoclonal antibody against CD45 or CD34 (eBioscience, San Diego, CA, USA) for 30 min at 4 °C in the dark. Flow cytometry analysis was performed (Beckman XL, Beckman Coulter, Brea, CA, USA).

Mol Cell Biochem

RT-PCR

Table 1 Treatment groups Groups

Influence factors

Control (CON)

10 % FBS only

IL-2

Interleukin-2 (20 ng/ml)

IL-6

Interleukin-6 (20 ng/ml)

TNF-a

Tumor necrosis factor-a (20 ng/ml)

SCF

Stem cell factor (50 ng/ml)

LPS

Lipopolysaccharides (10 lg/ml)

Mixture of cytokines, SCF and LPS (CM)

IL-2 (20 ng/ml), IL-6 (20 ng/ml), LPS (10 lg/ml), TNF-a (20 ng/ml) and SCF (50 ng/ml)

Serum from rat with ALF (LFS)

10 % serum from SD rats with acute liver failure

Serum from healthy rats (NS) (n = 10)

10 % serum from healthy SD rats

FBS fetal bovine serum, SD Sprague–Dawley, ALF acute liver failure

Adipogenic and osteogenic differentiation For adipogenic induction, rUMSCs at passage 3 were plated at a density of 1 9 104 cells/cm2 in adipogenic induction medium (Cyagen Biosciences Inc., Santa Clara, CA, USA). After 21 days, lipid vesicles were revealed by oil-red O (Cyagen Biosciences Inc., Santa Clara, CA, USA) staining for 30 min (Supplementary Fig. 3A). For osteogenic induction, rUMSCs were plated at a density of 5 9 103 cells/cm2 in osteogenic induction medium (Cyagen Biosciences Inc., Santa Clara, CA, USA). After 21 days, calcium deposition in rUMSCs was evaluated by alizarin red solution (Cyagen Biosciences Inc., Santa Clara, CA, USA) for 5 min (Supplementary Fig. 3B). Rat model of acute liver failure ALF model was induced in 10 male SD rats as previously described [25, 26]. D-galactosamine (D-GalN, Toronto Research Chemicals, Toronto, Canada) (1.4 g/kg) was injected intraperitoneally to induce ALF. After D-galactosamine treatment for 12 h, serum obtained from rats with ALF (LFS) was preserved for further use. Pathological tissue degeneration and necrosis were assessed to determine the success of the modeling. Treatment of rUMSCs with various factors rUMSCs at passage 3 were plated at a density of 2 9 104 cells/cm2 and cultured in growth medium. After 24 h, cells were cultured for 48 h in medium containing cytokines, lipopolysaccharide (LPS), stem cell factor (SCF), or LFS (Table 1). After incubation, cells were harvested and used for different assays to analyze CXCR4 expression.

Expression of CXCR4 mRNA was analyzed by RT-PCR. Total RNA extraction (RNeasy Mini Kit, Qiagen, Venlo, Netherlands) and reverse transcription (Omniscript RT Kit, Qiagen, Venlo, Netherlands) were performed according to the manufacturer’s instructions. CXCR4 primers were 50 AGC CTG GAC CGC TAC CTT-30 (forward) and 50 -ATG ATG TGC TGG AAC TGG-30 (reverse) (Product length: 224 bp). b-actin primers were 50 -TCC TGT GGC ATC CAC GAA ACT-30 (forward) and 50 -GAA GCA TTT GCG GTG GAC GAT-30 (reverse) (Product length: 315 bp). PCR protocol was: initial denaturation at 95 °C for 15 min, followed by 35 cycles at 94 °C for 45 s, 58 °C for 1 min, 72 °C for 45 s, and final extension at 72° for 10 min. PCR products were separated on 2 % agarose gels, and scanned with a Gel Imaging System (Bio-Rad, Hercules, CA, USA). The amount of CXCR4 mRNA was quantified using the Scion Image 4.03 software (Scion Corporation, Frederick, MD, USA) and expressed relatively to b-actin. Western blot Expression of CXCR4 protein was detected by western blot. Cells were washed with PBS and lysed with 1 ml/ well of lysis buffer (Beyotime, Jiangsu, China) containing 20 mM of Tris, 150 mM of NaCl, 1 mM of EDTA, 1 % Triton X-100, 2.5 mM of sodium pyrophosphate, 1 mM of glycerolphosphate, 1 mM of Na3VO4, 1 g/ml of leupeptin, and 1 mM of phenylmethylsulfonyl fluoride (PMSF), pH7.5, for 30 min at 4 °C. The extract was centrifuged at 12,0009g at 4 °C for 10 min, and the protein concentration was determined using a BCA kit (Pierce Chemical, Dallas, TX, USA). Proteins (30 lg) were separated by 10 % SDS-PAGE and transferred onto PVDF membranes (Bio-Rad, Hercules, CA, USA). Non-specific sites were blocked with 5 % skim milk powder diluted in TBS with 0.05 % Tween 20 (TBST) for 2 h. Western blot was performed using rabbit anti-CXCR4 polyclonal antibody (BioVision Inc., Milpitas, CA, USA) and rabbit monoclonal anti-b-actin (LianKe Biological Technology Co., LTD, Hangzhou, China) at 4 °C, overnight. Bands were visualized using HRP-labeled goat anti-rabbit IgG (Beyotime, Jiangsu, China), and ECL western blot detection reagents (Biological Industries, Kibbutz Beit-Haemek, Israel). The amount of protein was semi-quantified using the Scion Image 4.03 software (Scion Corporation, Frederick, MD, USA). Transplantation of rUMSCs Thirty SD rats were randomly divided into 3 groups (10 rats/ group): in group A, rUMSCs were pretreated with LFS to

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Fig. 1 Effects of serum from rat with ALF and cytokines on CXCR4 mRNA expression in rUMSCs at the third passage. mRNA expression was determined by semi-quantitative RT-PCR. b-actin was used as an internal control. Data are shown as mean ± standard deviation (SD). a P \ 0.05 versus the CON group; b P \ 0.05 versus the NS group. CXCR4 C-X-C chemokine receptor type 4, TNF-a tumor necrosis factora, CM mixture of cytokines, SCF and LPS, SCF stem cell factor, NS serum from healthy rat, ALF serum from rat with acute liver failure, LPS lipopolysaccharide, IL-6 interleukin-6, IL-2 interleukin-2, CON rUMSCs treated with only containing 10 % FBS

enhance CXCR4 expression before injection; in group B, rUMSCs were not pretreated with any factors prior to injection; and in group C, normal saline solution (1 ml) was directly injected in the jugular vein, without any rUSMCs. Before transplantation, rUMSCs were adjusted to a concentration of 1 9 107/ml and labeled with carboxyfluorescein (CFSE, 0.03 lg/ml) (Dojindo Molecular Technologies, Kimamoto, Japan) at 37 °C for 5–10 min. After washing with PBS, CFSE-labeled rUMSCs (3 9 106 cells in 1 ml PBS) were slowly injected in the jugular vein of male rats. After rUMSCs transplantation for 12 h, D-GalN (1.4 g/kg) was injected intraperitoneally to induce ALF. Rats from each group were killed 48 h or 1 week after transplantation. Livers were removed, and frozen sections were prepared. CFSE-labeled rUMSCs in liver tissues were analyzed under fluorescence microscopy (Nikon, Tokyo, Japan).

Statistical analysis All statistical analyses were conducted using Prism v5.0 (GraphPad Software, San Diego, CA, USA). Data are

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Fig. 2 Effects of serum from rat with ALF and cytokines on CXCR4 protein expression in rUMSCs at the third passage. Protein expression was determined by western blot. b-actin was used as an internal control. Data are shown as mean ± SD. a P \ 0.05 versus the CON group; b P \ 0.05 versus the NS group

presented as mean ± standard deviation (SD) for at least three sets of independent experiments. Differences between groups were assessed using one-way analysis of variance (ANOVA) with the Tukey’s post hoc test. Differences were considered to be statistically significant when P \ 0.05.

Results LFS and cytokines enhanced the expression of CXCR4 in rUMSCs The relative expression of CXCR4 mRNA in the control group and serum from the healthy rats (NS) group was 51.13 ± 9.63 and 52.83 ± 9.85 %, respectively (P = 0.841). The relative expression of CXCR4 mRNA in the IL-2, IL-6, LPS, LFS, CM, SCF, and TNF-a groups was 88.26 ± 17.16 % (1.73-fold), 83.16 ± 13.56 % (1.63-fold), 79.03 ± 13.81 % (1.55-fold), 118.06 ± 22.83 % (2.31-fold), 112.51 ± 28.27 % (2.20-fold), 102.93 ± 26.71 % (2.01-fold), and 73.79 ± 9.50 % (1.44-fold), respectively (all P \ 0.05, compared with the control group; IL-2, IL-6, LFS, CM and SCP groups: P \ 0.05 vs. NS). CXCR4 mRNA expression was strongly induced by LFS and CM, respectively (Fig. 1). Compared with the control group, CXCR4 protein levels in the IL-2, IL-6, LPS, LFS, CM, SCF, and TNF-a groups were 46.32 ± 12.21 % (2.06-fold), 41.77 ± 10.65 %

Mol Cell Biochem Fig. 3 Effects of serum from rat with ALF and cytokines on expression of cell surface CXCR4 in rUMSCs at the third passage, by flow cytometry. FITC-conjugated secondary antibody was used as negative control (isotype control). Data are shown as mean ± SD. a P \ 0.05 versus the CON group; b P \ 0.05 versus the NS group; c P \ 0.05 versus IL2 group; d P \ 0.05 versus IL-6 group; e P \ 0.05 versus LPS group; f P \ 0.05 versus LFS group; g P \ 0.05 versus CM group

(1.86-fold), 41.01 ± 8.02 % (1.83-fold), 58.56 ± 13.94 % (2.61-fold), 55.48 ± 12.96 % (2.47-fold), 51.17 ± 11.01 % (2.28-fold), and 40.94 ± 9.46 % (1.82-fold), respectively (all P \ 0.05, compared with the control group; IL-2, LPS, LFS, CM, and SCF groups: P \ 0.5 vs. NS). There was no difference between the NS and control groups (22.44 ± 6.81 vs. 18.48 ± 5.10 %, respectively, P = 0.466). CXCR4 protein levels in the SCF and IL-2 groups were higher than that in the IL-6, LPS, and TNF-a groups, but there were no significant difference (P [ 0.05). CXCR4 protein levels were strongly up-regulated by LFS and CM, respectively (Fig. 2).

The expression of cell surface CXCR4 on rUMSCs was analyzed by flow cytometry. The proportion of CXCR4positive cells in the untreated and in the NS groups was low (9.67 ± 1.06 and 9.7 ± 1.3 %, respectively, P = 0.974). The proportions of CXCR4-positive cells in groups treated with IL-2, IL-6, LPS, LFS, CM, SCF, and TNF-a were higher than that in the control group, i.e., 27.83 ± 1.89 % (2.88-fold), 22.40 ± 1.81 % (2.32-fold), 19.07 ± 1.17 % (1.97-fold), 42.37 ± 1.60 % (4.38-fold), 40.17 ± 1.78 % (4.15-fold), 31.40 ± 1.61 % (3.25-fold), and 16.37 ± 1.75 % (1.69-fold), respectively (all P \ 0.05, compared with the control and NS groups). The proportion of

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Mol Cell Biochem Fig. 4 Frozen sections of liver tissue from recipients at 48 h and 1 week after transplantation under fluorescence microscopy (bar: 20 lm). a1 48 h after transplantation in group A, rUMSCs emerged at periportal area. a2 1 week after transplantation in group A, rUMSCs migrated to hepatic cords of injured area. b1 48 h after transplantation in group B, rUMSCs also emerged at periportal area. The number of CSF-labeled rUMSCs was less than that in group A. (arrows: CFSE-labeled rUMSCs). b2 1 week after transplantation in group B, rUMSCs migrated to hepatic cords of injured area. In group C, normal saline solution (1 ml) was directly injected in the jugular vein, without any rUSMCs. c1 48 h after transplantation in the control group, no rUMSCs was observed. c2 1 week after transplantation in the control group, no rUMSCs was observed

CXCR4-positive cells in the SCF and IL-2 groups was significantly higher than that in the IL-6, LPS, and TNF-a groups. Serum from rats with ALF and CM strongly induced the surface expression of CXCR4 on rUMSCs (all P \ 0.001) (Fig. 3)

Fig. 4a2) than that in group B (in which rUMSCs were untreated before transplantation; Fig. 4b2). No rUMSCs was observed in the control group (Fig. 4c1, c2). Up-regulation of CXCR4 in rUMSCs induced by LFS was correlated with the improved homing capacities of rUMSCs to injured liver tissue.

Up-regulation of CXCR4 increased the homing of rUMSCS to damaged liver tissue Discussion CFSE-labeled rUMSCs were injected through the jugular vein into rats with D-GalN-induced ALF to assess recruitment of transplanted cells to damaged liver tissue. At 48 h after transplantation, the recipients were sacrificed. Frozen sections of liver tissue were analyzed by fluorescence microscopy [27]. In groups A and B, CSFE-labeled rUMSCs were observed mainly in the portal area 48 h after transplantation (Fig. 4a1, b1). At 1 week after transplantation, rUMSCs migrated to hepatic cords in injured area. There was more rUMSCs homing to damaged liver tissue in group A (in which rUMSCs were pretreated with LFS;

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The liver is the only organ capable of regeneration in response to acute injury, which does not rely on stem cells or progenitor cells, but instead involves mitosis of mature hepatocytes [28]. MSCs have the capacity to differentiate into hepatocyte-like cells [4–6]. The aim of the present study was to evaluate CXCR4 expression in rUMSCs, and their homing to injured liver tissue. rUMSCs were successfully isolated (Supplementary Fig. 1), expressing CD90 and CD106, but not CD34 and CD45 (Supplementary Fig. 2). mRNA and protein expressions of CXCR4

Mol Cell Biochem

were strongly up-regulated by LFS and by the mixture of cytokines, SCF, and LPS (CM). Expression of cell surface CXCR4 on rUMSCs in groups treated with LFS and CM was higher than that in the untreated control group. At 48 h after transplantation, more rUMSCs pretreated with LFS appeared in the portal area, and migrated to the liver parenchyma after 1 week. These results could eventually lead to new ALF treatments to overcome donor shortage. A major impairment of previous studies is the use of human USMCs in animal models. Indeed, there are a number of studies about the effects of human UMSCs transplantation into immunosuppressed or nude rats models of a number of diseases [4, 14, 20, 21, 29–31]. These studies provide useful insights into homing and repair mechanisms, but xenogeneic transplantation cannot emulate the real clinical situation and its efficacy is not very good. In addition, among others, there are differences in SDF-1 and CXCR4 between humans and rats [4, 14, 20, 21, 29, 30, 32]. Indeed, studies using MSC transplantation within the same species showed better efficacy [6, 22, 23, 33]. Therefore, we successfully isolated UMSCs from the umbilical cords of SD rats using the two-step digestion method using collagenase and trypsin [24]. MSCs have no specific marker; they are identified solely based on the lack of hematopoietic markers and the expression of non-specific mesenchymal markers, plastic adherence, and potential for multiple differentiations (in the present study, adipocytes and osteocytes). These characteristics are considered necessary and sufficient to characterize MSCs [34]. Therefore, rUMSCs isolated in the present study were consistent with MSCs’ characteristics by their morphology, markers, and multiple differentiation potential [8, 22, 24, 32]. It was reported that MSCs express mesenchymal markers such as CD29, CD44, CD71, CD73, CD90, CD105, SH-2, and SH-3, and that they lack hematopoietic markers such as CD45, CD34, CD133, CD11b, MHCII, and F4/80 [35, 36]. In the present study, rUMSCs at passage 3 were strongly positive for the MSC marker CD90. CD106 was expressed only in 25.2 % of the population. rUMSCs, just as human UMSCs, were negative for hematopoietic markers CD34 and CD45 [35, 36]. Therefore, we used rUMSCs at passage 3, as previously described [18, 19], and also because CXCR4 expression tends to decrease with high number of passages [11], while it is stable at passages 1–8 [17]. Previous studies showed that transplantation of MSCs from human umbilical cord blood could significantly improve the survival of rats with acute hepatic necrosis [4, 7, 8, 32]. The underlying mechanisms involved may include the transdifferentiation of MSCs into hepatocytelike cells and targeted migration of these cells to liver lesion sites [32]. However, transplantation efficiency was low, only reaching 0.49 % of total liver cell mass [4].

Single injection of high cell numbers into the portal vein is difficult as this result in an increased mortality as MSCs are probably massively entrapped in the lung and provoke pulmonary artery thrombosis [27]. Furthermore, even if MSCs have a low immunogenic potential and transdifferentiate into cells that also have a low immunogenic potential [8], the difference in species could contribute to the low efficacy [4, 7, 8, 32]. Therefore, the key to increase transplantation efficiency is to use MSCs from the same species and to enhance homing of MSCs to injured lesion of the liver. In the present study, CXCR4 levels on the cell surface of MSCs were low after being cultured in vitro, as previously observed [17]. Increasing CXCR4 expression might be a potential strategy to improve engraftment of MSCs in bone marrow and accelerate hematopoiesis recovery [14]. Previously, we observed that CXCR4 on MSCs were up-regulated by cytokines, and that SDF-1 was highly expressed on biliary epithelial cells in injured liver tissue [37, 38]. However, we acknowledge that even if CXCR4 plays an important role in cell homing, it is not the only factor involved [15, 16]. Further studies are required to assess these other factors, and to assess their relationship. It was reported that short-term stimulation with a cytokine cocktail resulted in up-regulation of both cell surface and intracellular CXCR4 in human MSCs, increasing their in vitro migration capacity to SDF-1 and homing to the bone marrow of irradiated NOD/SCID mice [14]. In ALF, the inflammatory and body balance are heavily disturbed by cytokines such as IL-2, IL-6, and TNF-a. Indeed, IL-2 is produced by T cells during an immune response, and stimulates the growth, proliferation, and differentiation of a number of cell types involved in immunity [39]. IL-6 secreted by T cells and macrophages stimulate the immune response during infection and after trauma, especially in tissue damage leading to inflammation [40]. In the same way, TNF-a plays a number of roles in the acute inflammatory phase. It can also activate the NF-jB and the MAPK pathways, both involved in cell survival, proliferation, and differentiation [41]. SCF is critical for the selfrenewal and proliferation of MSCs [38]. Therefore, we selected IL-2, IL-6, LPS, TNF-a, SCF, a cocktail of cytokines, LPS, and SCF, and LFS to study their influence on CXCR4 expression in rUMSCs. Our results showed that CXCR4 expression at the mRNA and protein levels was up-regulated by IL-2, IL-6, TNF-a, SCF, LPS, and LFS. Consequently, we observed that the expression of CXCR4 on rUMSCs membrane was also up-regulated by these factors. Taken independently, the effects of these cytokines on rUSMCs are consistent with their roles in the response to an injury, i.e., promoting the survival, proliferation, and differentiation of cell types involved in this response. Classical models showed that immune cells, such

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as T-cells and dendritic cells, are the main targets of these cytokines [39–41]. However, our study also showed that USMCs respond to these cytokines since the CXCR4 mRNA and protein levels are increased after treatment using these cytokines. These results suggest that USMCs are part of the response system against an injury. Because the present study is a preclinical one, we chose not to use an overexpression system in order to remain in a clinical setting. Instead, serum from an ALF patient could be used to stimulate its own stem cells to express more CXCR4. We used ALF serum to induce CXCR4 expression on rUMSCs membrane. In group A, the proportion of CXCR4-positive cells was increased after rUMSCs were treated with ALF, but this proportion remained low in untreated cells. Our data showed that rUMSCs appeared in the portal areas 48 h after transplantation and migrated to hepatic cords in injured area. More rUMSCs homed to damaged liver tissue in group A. Up-regulation of CXCR4 induced by ALF enhanced homing of rUMSCs to liver tissue. Consistently with our results, Shi et al. [32] also reported that human UMSCs dispersed at portal area at 48 h after transplantation to rat model with acute hepatic necrosis. Human UMSCs were found in necrotic liver areas 1 week later and persisted after 4 weeks [32]. The 48-hour survival rate after transplantation was up to 80 % in group A, 70 % in group B, and 40 % in group C (data not shown). Therefore, rUMSCs might have therapeutic effects on ALF. Enhancing homing of rUMSCs may improve survival of ALF rats. However, the mechanisms underlying this effect are unclear. First, rUMSCs may directly transdifferentiate into hepatocytes. Secondly, MSC-secreted factors might inhibit apoptosis, stimulate hepatocyte regeneration, and minimize inflammatory infiltration in the liver around the MSCs [42, 43]. Future studies should aim toward understanding these mechanisms. The present study suffered some limitations. First, we did not assess mid- and long-term rat survival after transplantation. This issue is important, and will be addressed in future studies. Second, the long-term effect of MSCs is unclear (such as malignancy potential), and studies should also assess this point. Third, although it was reported that UMSCs can be induced into hepatocytes in vitro [44, 45], it is still unknown if these cells really transdifferentiate into hepatocytes in vivo. Finally, an animal model is never perfect; ALF was chemically induced, and we cannot exclude some remaining effects of the chemical, or effects in other organs. There is a long way to go until UMSCs can be used for clinical application. However, results of the present study offer an additional insight for reaching success. In conclusion, we successfully isolated rUMSCs with multipotential differentiation from rats’ umbilical cords.

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CXCR4 expressed in rUMSCs, which was up-regulated by cytokines, LPS, SCF, and LFS. Increasing CXCR4 expression on the membrane of rUMSCs may enhance their homing to injured liver tissue. Acknowledgments This study was supported by the Social Development Program of Jiangsu Province (BE2010650) and Science of Jiangsu Province (BK20130271).

References 1. Allameh A, Kazemnejad S (2012) Safety evaluation of stem cells used for clinical cell therapy in chronic liver diseases; with emphasize on biochemical markers. Clin Biochem 45:385–396. doi:10.1016/j.clinbiochem.2012.01.017 2. Soto-Gutierrez A, Navarro-Alvarez N, Yagi H, Yarmush ML (2009) Stem cells for liver repopulation. Curr Opin Organ Transplant 14:667–673. doi:10.1097/MOT.0b013e3283328070 3. Wu YM, Joseph B, Berishvili E, Kumaran V, Gupta S (2008) Hepatocyte transplantation and drug-induced perturbations in liver cell compartments. Hepatology 47:279–287. doi:10.1002/ hep.21937 4. Sato Y, Araki H, Kato J, Nakamura K, Kawano Y, Kobune M, Sato T, Miyanishi K, Takayama T, Takahashi M, Takimoto R, Iyama S, Matsunaga T, Ohtani S, Matsuura A, Hamada H, Niitsu Y (2005) Human mesenchymal stem cells xenografted directly to rat liver are differentiated into human hepatocytes without fusion. Blood 106:756–763. doi:10.1182/blood-2005-02-0572 5. Wu XB, Tao R (2012) Hepatocyte differentiation of mesenchymal stem cells. Hepatobiliary Pancreat Dis Int 11:360–371 6. Stock P, Bruckner S, Ebensing S, Hempel M, Dollinger MM, Christ B (2010) The generation of hepatocytes from mesenchymal stem cells and engraftment into murine liver. Nat Protoc 5:617–627. doi:10.1038/nprot.2010.7 7. Yan JQ, Han T, Zhu ZY (2008) BiologicaI characteristics and differentiation into hepatocyte-like cells of human umbilical cord-derived mesenchymal stem Cells. Shijie Huaren Xiaohua Zazhi 16:1639–1644 8. Zhao Q, Ren H, Li X, Chen Z, Zhang X, Gong W, Liu Y, Pang T, Han ZC (2009) Differentiation of human umbilical cord mesenchymal stromal cells into low immunogenic hepatocyte-like cells. Cytotherapy 11:414–426. doi:10.1080/14653240902849754 9. Cho KA, Ju SY, Cho SJ, Jung YJ, Woo SY, Seoh JY, Han HS, Ryu KH (2009) Mesenchymal stem cells showed the highest potential for the regeneration of injured liver tissue compared with other subpopulations of the bone marrow. Cell Biol Int 33:772–777. doi:10.1016/j.cellbi.2009.04.023 10. Bhakta S, Hong P, Koc O (2006) The surface adhesion molecule CXCR4 stimulates mesenchymal stem cell migration to stromal cell-derived factor-1 in vitro but does not decrease apoptosis under serum deprivation. Cardiovasc Revasc Med 7:19–24. doi:10.1016/j.carrev.2005.10.008 11. Son BR, Marquez-Curtis LA, Kucia M, Wysoczynski M, Turner AR, Ratajczak J, Ratajczak MZ, Janowska-Wieczorek A (2006) Migration of bone marrow and cord blood mesenchymal stem cells in vitro is regulated by stromal-derived factor-1-CXCR4 and hepatocyte growth factor-c-met axes and involves matrix metalloproteinases. Stem Cells 24:1254–1264. doi:10.1634/stem cells.2005-0271 12. Honczarenko M, Le Y, Swierkowski M, Ghiran I, Glodek AM, Silberstein LE (2006) Human bone marrow stromal cells express a distinct set of biologically functional chemokine receptors. Stem Cells 24:1030–1041. doi:10.1634/stemcells.2005-0319

Mol Cell Biochem 13. Beider K, Nagler A, Wald O, Franitza S, Dagan-Berger M, Wald H, Giladi H, Brocke S, Hanna J, Mandelboim O, Darash-Yahana M, Galun E, Peled A (2003) Involvement of CXCR4 and IL-2 in the homing and retention of human NK and NK T cells to the bone marrow and spleen of NOD/SCID mice. Blood 102:1951–1958. doi:10.1182/blood-2002-10-3293 14. Shi M, Li J, Liao L, Chen B, Li B, Chen L, Jia H, Zhao RC (2007) Regulation of CXCR4 expression in human mesenchymal stem cells by cytokine treatment: role in homing efficiency in NOD/SCID mice. Haematologica 92:897–904 15. Lapidot T, Kollet O (2002) The essential roles of the chemokine SDF-1 and its receptor CXCR4 in human stem cell homing and repopulation of transplanted immune-deficient NOD/SCID and NOD/SCID/B2 m(null) mice. Leukemia 16:1992–2003. doi:10. 1038/sj.leu.2402684 16. Kaifi JT, Yekebas EF, Schurr P, Obonyo D, Wachowiak R, Busch P, Heinecke A, Pantel K, Izbicki JR (2005) Tumor-cell homing to lymph nodes and bone marrow and CXCR4 expression in esophageal cancer. J Natl Cancer Inst 97:1840–1847. doi:10. 1093/jnci/dji431 17. Askari AT, Unzek S, Popovic ZB, Goldman CK, Forudi F, Kiedrowski M, Rovner A, Ellis SG, Thomas JD, DiCorleto PE, Topol EJ, Penn MS (2003) Effect of stromal-cell-derived factor 1 on stem-cell homing and tissue regeneration in ischaemic cardiomyopathy. Lancet 362:697–703. doi:10.1016/S01406736(03)14232-8 18. Broxmeyer HE, Orschell CM, Clapp DW, Hangoc G, Cooper S, Plett PA, Liles WC, Li X, Graham-Evans B, Campbell TB, Calandra G, Bridger G, Dale DC, Srour EF (2005) Rapid mobilization of murine and human hematopoietic stem and progenitor cells with AMD3100, a CXCR4 antagonist. J Exp Med 201:1307–1318. doi:10.1084/jem.20041385 19. Kyriakou C, Rabin N, Pizzey A, Nathwani A, Yong K (2008) Factors that influence short-term homing of human bone marrowderived mesenchymal stem cells in a xenogeneic animal model. Haematologica 93:1457–1465. doi:10.3324/haematol.12553 20. Aurich I, Mueller LP, Aurich H, Luetzkendorf J, Tisljar K, Dollinger MM, Schormann W, Walldorf J, Hengstler JG, Fleig WE, Christ B (2007) Functional integration of hepatocytes derived from human mesenchymal stem cells into mouse livers. Gut 56:405–415. doi:10.1136/gut.2005.090050 21. Vilalta M, Degano IR, Bago J, Gould D, Santos M, Garcia-Arranz M, Ayats R, Fuster C, Chernajovsky Y, Garcia-Olmo D, Rubio N, Blanco J (2008) Biodistribution, long-term survival, and safety of human adipose tissue-derived mesenchymal stem cells transplanted in nude mice by high sensitivity non-invasive bioluminescence imaging. Stem Cells Dev 17:993–1003. doi:10.1089/ scd.2007.0201 22. Ganta C, Chiyo D, Ayuzawa R, Rachakatla R, Pyle M, Andrews G, Weiss M, Tamura M, Troyer D (2009) Rat umbilical cord stem cells completely abolish rat mammary carcinomas with no evidence of metastasis or recurrence 100 days post-tumor cell inoculation. Cancer Res 69:1815–1820. doi:10.1158/0008-5472. CAN-08-2750 23. Wang S, Zhou C, Qin AL (2012) Abstract #558—The therapeutic potential of rat umbilical mesenchymal stem cells from Warton’s jelly in the treatment of rat chronic pancreatitis and subsequent pancreatic fibrosis induced by DBTC. Gastroenterology 142:S112–S113 24. Liu KL, Shi BY, Liu DZ, Jin JG, Li HB, Shi YC, Feng K, Xiao L (2010) Isolation and biological characteristics of rat umbilical cord mesenchymal stem cells Modern. Rehabilitation 14:1743–1748 25. Zhang L, Kang W, Lei Y, Han Q, Zhang G, Lv Y, Li Z, Lou S, Liu Z (2011) Granulocyte colony-stimulating factor treatment ameliorates liver injury and improves survival in rats with D-

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

40.

41.

galactosamine-induced acute liver failure. Toxicol Lett 204:92–99. doi:10.1016/j.toxlet.2011.04.016 Zhang Y, Hu XY, Luo JX, Chen G, Gao SD (2012) Effect of herbs for clearing heat and resolving stasis on liver function and survival in rats with acute liver failure. Shijie Huaren Xiaohua Zazhi 20:1961–1966 Schrepfer S, Deuse T, Reichenspurner H, Fischbein MP, Robbins RC, Pelletier MP (2007) Stem cell transplantation: the lung barrier. Transpl Proc 39:573–576. doi:10.1016/j.transproceed. 2006.12.019 Fausto N, Campbell JS (2003) The role of hepatocytes and oval cells in liver regeneration and repopulation. Mech Dev 120:117–130 Yang CC, Shih YH, Ko MH, Hsu SY, Cheng H, Fu YS (2008) Transplantation of human umbilical mesenchymal stem cells from Wharton’s jelly after complete transection of the rat spinal cord. PLoS ONE 3:e3336. doi:10.1371/journal.pone.0003336 Tsai PC, Fu TW, Chen YM, Ko TL, Chen TH, Shih YH, Hung SC, Fu YS (2009) The therapeutic potential of human umbilical mesenchymal stem cells from Wharton’s jelly in the treatment of rat liver fibrosis. Liver Transpl 15:484–495. doi:10.1002/lt.21715 Hong SQ, Zhang HT, You J, Zhang MY, Cai YQ, Jiang XD, Xu RX (2011) Comparison of transdifferentiated and untransdifferentiated human umbilical mesenchymal stem cells in rats after traumatic brain injury. Neurochem Res 36:2391–2400. doi:10. 1007/s11064-011-0567-2 Shi LL, Liu FP, Wang DW (2011) Transplantation of human umbilical cord blood mesenchymal stem cells improves survival rates in a rat model of acute hepatic necrosis. Am J Med Sci 342:212–217. doi:10.1097/MAJ.0b013e3182112b90 Sgodda M, Aurich H, Kleist S, Aurich I, Konig S, Dollinger MM, Fleig WE, Christ B (2007) Hepatocyte differentiation of mesenchymal stem cells from rat peritoneal adipose tissue in vitro and in vivo. Exp Cell Res 313:2875–2886. doi:10.1016/j.yexcr.2007. 05.020 Christ B, Dollinger MM (2011) The generation of hepatocytes from mesenchymal stem cells and engraftment into the liver. Curr Opin Organ Transpl 16:69–75. doi:10.1097/MOT. 0b013e3283424f5b Kim JW, Kim SY, Park SY, Kim YM, Kim JM, Lee MH, Ryu HM (2004) Mesenchymal progenitor cells in the human umbilical cord. Ann Hematol 83:733–738. doi:10.1007/s00277-004-0918-z Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, Deans R, Keating A, Prockop D, Horwitz E (2006) Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society For Cellular Therapy position statement. Cytotherapy 8:315–317. doi:10.1080/ 14653240600855905 Huang XP, Qin AL, Zhao WF (2009) CXCR4 expression on the surface of bone marrow mesenchymal stem cells in chronic liver failure environment and the influential factors. Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 13:7885–7888 Lee Y, Jung J, Cho KJ, Lee SK, Park JW, Oh IH, Kim GJ (2013) Increased SCF/c-kit by hypoxia promotes autophagy of human placental chorionic plate-derived mesenchymal stem cells via regulating the phosphorylation of mTOR. J Cell Biochem 114:79–88. doi:10.1002/jcb.24303 Lan RY, Selmi C, Gershwin ME (2008) The regulatory, inflammatory, and T cell programming roles of interleukin-2 (IL-2). J Autoimmun 31:7–12. doi:10.1016/j.jaut.2008.03.002 van der Poll T, Keogh CV, Guirao X, Buurman WA, Kopf M, Lowry SF (1997) Interleukin-6 gene-deficient mice show impaired defense against pneumococcal pneumonia. J Infect Dis 176:439–444 Kant S, Swat W, Zhang S, Zhang ZY, Neel BG, Flavell RA, Davis RJ (2011) TNF-stimulated MAP kinase activation

123

Mol Cell Biochem mediated by a Rho family GTPase signaling pathway. Genes Dev 25:2069–2078. doi:10.1101/gad.17224711 42. van Poll D, Parekkadan B, Cho CH, Berthiaume F, Nahmias Y, Tilles AW, Yarmush ML (2008) Mesenchymal stem cell-derived molecules directly modulate hepatocellular death and regeneration in vitro and in vivo. Hepatology 47:1634–1643. doi:10.1002/ hep.22236 43. Parekkadan B, van Poll D, Suganuma K, Carter EA, Berthiaume F, Tilles AW, Yarmush ML (2007) Mesenchymal stem cellderived molecules reverse fulminant hepatic failure. PLoS ONE 2:e941. doi:10.1371/journal.pone.0000941

123

44. Lee KD, Kuo TK, Whang-Peng J, Chung YF, Lin CT, Chou SH, Chen JR, Chen YP, Lee OK (2004) In vitro hepatic differentiation of human mesenchymal stem cells. Hepatology 40:1275–1284. doi:10.1002/hep.20469 45. Zhang YN, Lie PC, Wei X (2009) Differentiation of mesenchymal stromal cells derived from umbilical cord Wharton’s jelly into hepatocyte-like cells. Cytotherapy 11:548–558. doi:10.1080/ 14653240903051533