Author Affiliations: The State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, First Affiliated Hospital, Zhejiang University School of Medicine; Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, 79 Qingchun Road, Hangzhou 310003, China (Yang JF, Cao HC, Pan QL, Yu J, Li J and Li LJ)

Corresponding Author: Professor Lan-Juan Li, MD, The State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, First Affiliated Hospital, Zhejiang University School of Medicine; Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, 79 Qingchun Road, Hangzhou 310003, China (Tel: +86-571-87236458; Fax: +86-571- 87236459; Email: ljli@zju.edu.cn)

 

© 2015, Hepatobiliary Pancreat Dis Int. All rights reserved.

doi: 10.1016/S1499-3872(15)60354-X

Published online March 18, 2015.

 

 

Acknowledgement: We thank the staff and the patients of Hangzhou Red Cross Hospital for providing the umbilical cord.

Contributors: YJF and CHC contributed equally to this article. YJF and CHC proposed the study, performed the research, and wrote the first draft. PQL, YJ and LJ collected and analyzed the data. All authors contributed to the design and interpretation of the study and to further drafts. LLJ is the guarantor.

Funding: This study was supported by grants from the National Natural Science Foundation of China (81471794), Chinese High-Tech Research & Development (863) Program (SS2013AA020102) and the National Science and Technology Major Project (2012ZX10002004).

Ethical approval: All procedures were approved by the Ethics Committee of Zhejiang University.

Competing interest: No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

 

 

BACKGROUND: Cell therapy has been promising for various diseases. We investigated whether transplantation of human umbilical cord mesenchymal stem cells (hUCMSCs) has any therapeutic effects on D-galactosamine/lipopolysaccharide (GalN/LPS)-induced fulminant hepatic failure in mice.

 

METHODS: hUCMSCs isolated from human umbilical cord were cultured and transplanted via the tail vein into severe combined immune deficiency mice with GalN/LPS-induced fulminant hepatic failure. After transplantation, the localization and differentiation of hUCMSCs in the injured livers were investigated by immunohistochemical and genetic analyses. The recovery of the injured livers was evaluated histologically. The survival rate of experimental animals was analyzed by the Kaplan-Meier method and log-rank test.

 

RESULTS: hUCMSCs expressed high levels of CD29, CD73, CD13, CD105 and CD90, but did not express CD31, CD79b, CD133, CD34, and CD45. Cultured hUCMSCs displayed adipogenic and osteogenic differentiation potential. Hematoxylin and eosin staining revealed that transplantation of hUCMSCs reduced hepatic necrosis and promoted liver regeneration. Transplantation of hUCMSCs prolonged the survival rate of mice with fulminant hepatic failure. Polymerase chain reaction for human alu sequences showed the presence of human cells in mouse livers. Positive staining for human albumin, human alpha-fetoprotein and human cytokeratin 18 suggested the formation of hUCMSCs-derived hepatocyte-like cells in vivo.

 

CONCLUSIONS: hUCMSC was a potential candidate for stem cell based therapies. After transplantation, hUCMSCs partially repaired hepatic damage induced by GalN/LPS in mice. hUCMSCs engrafted into the injured liver and differentiated into hepatocyte-like cells.

 

(Hepatobiliary Pancreat Dis Int 2015;14:186-193)

 

KEY WORDS: human umbilical cord mesenchymal stem cells; fulminant hepatic failure; cell transplantation; hepatic differentiation

 

Liver transplantation is by far the most effective way to treat fulminant hepatic failure (FHF). But the high cost and the shortage of donor liver severely limit its application. Cell-based approaches, such as hepatocyte transplantation, have been investigated in recent years.^[1,2] However, adult hepatocytes have limited proliferative potential and are insufficient to regenerate a damaged liver.^{[3, 4]} Recently, the transplantation of mesenchymal stem cells (MSCs) from various origins has been identified as an effective alternative therapy to treat different types of liver disease.^[5-8] MSCs are able to differentiate into hepatocyte-like cells in injured livers and thus, to restore liver function.^{[9, 10]}

 

Recently, we have successfully isolated MSCs from the human umbilical cord. These MSCs express human MSC-specific markers and can differentiate into various cell types.^[11-17] Human umbilical cord MSCs (hUCMSCs) have several advantages. First, hUCMSCs are easy to harvest with no appreciable ethical issues. Second, they possibly provide an inexhaustible, low-cost source of stem cells. Third, they are a subset of primitive stem cells with high plasticity and developmental flexibility.^[16] Forth, they are not tumorigenic and do not induce acute rejection reaction in vivo.^[16] These advantages make hUCMSCs a highly attractive alternative to hepatocytes for liver regenerative medicine. Preclinical experiments showed that hUCMSCs can be used in various animal disease models, such as intracerebral hemorrhage, cerebral ischemia, retinal disease, type I diabetes and spinal cord injuries. In all of these experiments, hUCMSCs successfully grafted into the recipient animals, differentiated into the corresponding cell lineages with corresponding functions.^[18]

 

The present study was to evaluate the therapeutic effect of hUCMSCs on D-galactosamine/lipopolysaccharide (GalN/LPS)-induced fulminant hepatic failure in mice.

 

 

Umbilical cord samples were collected from the donors after informed consent was obtained from Hangzhou Red Cross Hospital in China. The study protocol was approved by the Research Ethics Committee of the First Affiliated Hospital, Zhejiang University School of Medicine. The cords were rinsed twice with saline, dissected in culture plates and digested with 0.1% collagenase type IV (Invitrogen, USA). Filtered with a 100-µm strainer, mononuclear cells (MNCs) were obtained by Ficoll-Paque density gradient centrifugation (1.077 g/cm³) (Ficoll-Paque PLUS; GE Healthcare, Sweden). The MNCs were collected and cultured in MesenCult™ MSC Basal Medium (Human) with MesenCult™ Mesenchymal Stem Cell Stimulatory Supplements (STEMCELL Technologies Inc., Vancouver, Canada) at a density of 1×10⁶ cells/cm² in tissue culture flasks (Nunc Flasks, Denmark). The cells were allowed to adhere for 3-7 days, after which the medium was changed. After 80%-90% confluence was achieved, the cells were trypsinized with 0.25% trypsin/EDTA (Invitrogen) and subcultured for further expansion. The cultures were maintained at 37 �� in an incubator with humidified air at 5% CO₂.

 

For phenotyping of cell surface antigens, cells (at passage 3) were harvested, resuspended at a concentration of 1×10⁶ cells/100 µL, and incubated with mouse anti-human antibodies against CD13-phycoerythrin (PE), CD90-PE, CD133-PE, CD73-allophycocyanin (APC), CD31-APC, CD29-APC, CD105-APC, CD34-fluorescein isothiocyanate (FITC), CD79b-FITC, and CD45-TRI COLOR (PE-Cy5-PC5) (eBioscience Inc., San Diego, CA, USA). Labeled cells were assayed by Cytomics FC 500 MPL flow cytometry systems with MXP software (Beckman Coulter, Inc, Los Angeles, CA, USA).

 

Adipogenic differentiation was done by culturing hUCMSCs (at passage 3) in MesenCult™ Adipogenic Differentiation Kit (STEMCELL Technologies Inc.) according to the instructions. hUCMSCs cultured in growth medium were used as controls. Four weeks later, the cells were stained with Oil Red O to demonstrate adipogenic differentiation. Lipoprotein lipase (LPL) mRNA was also detected by reverse transcription-polymerase chain reaction (RT-PCR). Osteogenic differentiation was done by culturing hUCMSCs (at passage 3) in OriCell™ hMSC Osteogenic Differentiation Medium (Cyagen Biosciences, Guangzhou, China) according to the instructions. Four weeks later, osteogenic differentiation was evaluated by Alizarin Red S staining and osteopontin (OPN) mRNA detection. hUCMSCs cultured in growth medium were used as controls.

 

For RT-PCR, total RNA was extracted with the Trizol reagent (Invitrogen). The primers for LPL were: 5'-ATG GAG AGC AAA GCC CTG CTC-3', 5'-TAC AGG GCG GCC ACA AGT TTT-3'; for OPN were: 5'-CTA GGC ATC ACC TGT GCC ATA CC-3', 5'-CTA CTT AGA CTA CTT GAC CAG TGA C-3'; and for GAPDH were: 5'-GGG TCG GAA GGA TTC CTA-3', 5'-GGT CTC AAA CAT GAT CTG GG-3'. The PCR products were analyzed using agarose gel electrophoresis. Images were captured using Syngene GBox-HR Gel Doc System (Syngene, Cambridge, UK).

 

Male severe combined immune deficiency (SCID) mice (4-8 weeks old, 20-25 g) were purchased from the Laboratory Animal Center, Zhejiang University, China. All procedures were approved by the Animal Ethics Committee of Zhejiang University. The animals were maintained in a light- and temperature-controlled facility (25±1 �� with a 12-hour dark/light cycle).

 

To establish the FHF mouse model, a mixture of D-galactosamine (GalN) and lipopolysaccharide (LPS) (0.5 mg/g and 1 ng/g body weight, respectively, in 1 mL saline) was intraperitoneally injected into the mice. The mice were randomly divided into two groups: group A (n=13), the FHF mice received an intravenous infusion of 5×10⁵ MSCs in 500 µL DMEM via the tail vein immediately after the GalN/LPS induction. In group A, liver samples were collected from three mice at day 15 and they were not used for survival analysis; in group B (n=10), the FHF mice received the same volume of DMEM only. Liver samples from surviving animals were collected at day 15 (3 mice sacrificed) and day 30 (7 mice sacrificed) after hUCMSCs transplantation. Liver samples of dead animals were harvested by direct dissection. The liver tissues were either fixed in 10% formalin for histological and immunohistochemical analysis, or immediately frozen in liquid nitrogen and stored for molecular analysis.

 

Mouse serum levels of TNF-α were measured at day 1, 2 and 3 after GalN/LPS administration. For this assay, whole blood samples were collected in anti-coagulant free tube, incubated undisturbed at room temperature for 20 minutes, and centrifuged at 3 000 rpm for 10 minutes at 4 ��. Serum samples were stored at -80 ��. TNF-α level was assessed using ELISA kit for mouse TNF-α (Abcam, UK), following the manufacturer's instructions.

 

PCR was carried out to detect the transplanted hUCMSC-derived cells in the recipient mice livers. DNA extraction was done using a DNeasy® Blood & Tissue Kit (Qiagen, Hilden, Germany). Primers for alu sequences were 5'-CTG GGC GAC AGA ACG AGA TTC TAT-3', and 5'-CTC ACT ACT TGG TGA CAG GTT CA-3'. The PCR was done as follows: denature at 94 �� for 2 minutes, followed by amplification for 35 cycles of 94 �� for 30 seconds, 60 �� for 30 seconds, and 72 �� for 59 seconds. The PCR products were analyzed in the same way as mentioned above.

 

Liver samples were fixed with formalin, and embedded in paraffin. For histological examinations, 5-µm thick sections were stained with hematoxylin and eosin (HE). For immunohistochemical analyses, sections were heated in citrate buffer (0.02 mol/L, pH 5.8) for antigen retrieval. Non-specific binding was blocked by 5% bovine serum albumin in PBS. Then, samples were incubated with primary antibody against human alpha-fetoprotein (hAFP), human albumin (hALB), and human cytokeratin 18 (hCK18), as per the instructions. Endogenous peroxidase activity was prevented by immersion in 0.3% hydrogen peroxide in methanol for 15 minutes. After washing, the sections were incubated with horseradish peroxidase conjugated secondary antibody at 37 �� for 1 hour. Peroxidase activity was visualized by DAB Peroxidase Substrate Kit (Vector Labs, Burlingame, CA, USA). The sections were counterstained with hematoxylin and observed under a light microscope (TE2000; Nikon, Japan).

 

Data were presented as mean±standard deviation. Animal survival was analyzed by the Kaplan-Meier method and the log-rank test. The data were analyzed using the SPSS version 16.0 software (SPSS Inc., Chicago, IL, USA). P<0.05 was considered to be statistically significant.

 

 

Primary hUCMSCs formed colonies in 3 days after isolation (Fig. 1A). The cells at day 5 exhibited a fibroblastic morphology (Fig. 1B). Confluent cells at the first passage were arranged in parallel arrays (Fig. 1C).

 

The immunophenotypes of hUCMSCs were analyzed by flow cytometry. The cells expressed high levels of mesenchymal stem cell related markers including CD29, CD73, CD13, CD105 and CD90, and low levels of CD133, CD79b, CD31, CD45 and CD34 (Fig. 2). The lack of hematopoietic cell markers such as CD34 and CD45 confirmed that the hUCMSCs were not of hematopoietic origin. This phenotypic profile is consistent with MSCs, indicating that the cells are of the MSC lineage.

 

The hUCMSCs cultured in adipogenic medium showed the features of adipocytes in 4 weeks: differentiated hUCMSCs formed lipid droplets in cytoplasm which can be identified by Oil Red O staining, seen as multiple red vacuoles (Fig. 3B). The undifferentiated hUCMSCs contained no intracytoplasmic lipid droplets and showed negative staining (Fig. 3A). Adipogenic differentiation was further confirmed by LPL expression. After adipogenic induction, hUCMSCs expressed LPL mRNA. No LPL expression was found in hUCMSCs cultured in regular medium. hUCMSCs cultured in OriCell™ hMSC Osteogenic Differentiation Medium showed the characteristics of osteoblast: the extracellular calcium deposits in differentiated hUCMSCs was ascertained by Alizarin Red S staining (Fig. 3D). Osteogenic differentiation was further confirmed by OPN expression. After induction, hUCMSCs expressed OPN mRNA. These results were not seen in the hUCMSCs cultured in growth medium (Fig. 3C).

 

All of the 10 FHF animals died within 2 days in group B (six mice died within the first day after GalN/LPS induction); three mice died within 8 days in group A (survival time, 176±4.3 hours), three mice were sacrificed at day 15 and seven others were sacrificed at day 30. hUCMSCs transplantation immediately after the GalN/LPS injection significantly increased the survival rate (P<0.001) (Fig. 4A).

 

One day after GalN/LPS administration, serum TNF-α reached a high level of 828.18±9.78 pg/mL, followed by increasing levels at days 2 and 3 (850.56±28.88 and 894.54±12.46 pg/mL, respectively). However, TNF-α was undetectable in GalN/LPS-untreated mice (Fig. 4B).

 

To identify human-derived cells in the livers of recipient mice, human specific alu sequences were amplified by PCR. At day 8 post-transplantation, weak alu expression was seen in group A (Fig. 4C, lane 3), and this increased at day 15 (Fig. 4C, lane 4). Human alu sequences were expressed at a high level at day 30 (Fig. 4C, lane 5), demonstrating the successful implantation and expansion of transplanted human cells in the recipient FHF mice. There was no evidence of alu expression in group B (Fig. 4C, lane 1).

 

HE staining after GalN/LPS administration revealed seriously damaged lobular architecture in group B (Fig. 5A). The livers of FHF mice showed serious hepatic congestion, hepatocyte degeneration, disordered hepatocyte cords, as well as multiple and extensive areas of cellular necrosis and inflammatory cell infiltration. This liver damage was ameliorated after hUCMSCs treatment (Fig. 5B-D). There was less hepatic damage; fewer inflammatory cells and fewer necrotic hepatocytes in the treatment group. We also observed more binucleated cells with deeper cytoplasmic staining (Fig. 5B, C). This observation indicated that cell transplantation might promote the regeneration of damaged liver tissue. At 30 day post-transplantation, degeneration and necrosis were almost invisible, and a nearly normal lobular architecture was observed (Fig. 5D).

 

To investigate whether hUCMSCs had undergone hepatocyte differentiation in the liver, the expression of the hepatocyte-specific proteins hAFP, hALB and hCK18 was evaluated by immunohistochemistry. Representative immunohis tochemistry results are shown in Fig. 6. Eight days post-transplantation, the staining of hAFP, hALB and hCK18 was positive in liver specimens of recipient mice. At day 15, the number of cells staining positive for hAFP decreased, with a single spotty brown staining. However, the staining for hALB- and hCK18-positive cells was increased. At day 30, the number of hCK18-positive cells was continuously increased and appeared to form a cluster. There was no positive staining for all markers in the liver tissues from the untreated group.

 

 

MSCs, first described by Friedenstein in 1976,^[19] are defined by their high expression of CD73, CD90 and CD105 and lack of expression of CD11b, CD14, CD19, CD34, CD45, CD79a and HLA-DRs. Additionally, MSCs must be able to undergo chondrogenic, adipogenic and osteogenic differentiation in vitro.^[20] The youngest MSCs can be isolated from the umbilical cord. Our flow cytometry assay of cell surface antigens confirmed that the stem cells isolated from the umbilical cord had the characteristics of MSCs. They did not express hematopoietic cell antigens and but could differentiate into osteoblasts and adipocytes in vitro.

 

The GalN/LPS-induced FHF mouse model is a well-known experimental model and frequently used to study liver failure.^[21-23] Proinflammatory cytokines, including TNF-α, are considered to play an important role in the GalN/LPS-induced liver injury. We measured serum cytokine levels by ELISA and found that TNF-α levels were significantly increased in the GalN/LPS-treated mice. Histological examination showed that intraperitoneal injection of GalN/LPS induced serious liver damage including massive necrosis, inflammatory cell infiltration, and serious hemorrhage. hUCMSCs transplantation significantly attenuated liver damage in eight days with evidence of cell regeneration; HE staining showed nearly normal lobular structure at day 30. The 30-day survival rate was significantly higher in the hUCMSCs transplantation group than that in FHF mice with vehicle treatment (70% vs 0%). Our data demonstrated that hUCMSCs significantly ameliorated fulminant hepatic injury induced by GalN/LPS.

 

In cell-based therapies, the number of transplanted cells is a key factor. An appropriate cell dose is vital for the survival of the injured experimental animals. A dose of (2-10)×10⁶ MSCs per kg is used in most small animal experiments.^[24-27] In this study, we initially tested a dose of 1×10⁶ hUCMSCs in 0.5 mL DMEM. The recipient mice died within 12 hours (data not shown). When the cell dose was adjusted to 5×10⁵ hUCMSCs, we found that the mice can tolerant this dosage and there was no vein embolism.

 

The alu sequence can be used as a biomarker of human cells.^{[28, 29]} It is the most abundant human sequence and exists only in humans or other primates. The alu sequence was detected at different time points in the hUCMSCs group. This is consistent with previous reports that transplanted MSCs can migrate to injured organs.^[30-32] The differentiation of hUCMSCs into hepatocyte-like cells in the injured liver was examined by immunohistochemistry. Positive staining for human hepatic markers, including hAFP, hALB and hCK18, was observed in mouse livers at day 30 after hUCMSC transplantation. However, there was no positive staining for human hepatic markers in any of the liver samples from the vehicle group. The expression of human hepatocyte-specific markers suggested that hUCMSCs had migrated to the injured mouse livers and undergone hepatic differentiation. The mechanisms of the differentiation from hUCMSCs to liver cells need further investigation.

 

 

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