AJP - GI Journal of Applied Physiology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Gastrointest Liver Physiol 289: G1091-G1099, 2005. First published July 28, 2005; doi:10.1152/ajpgi.00049.2005
0193-1857/05 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
289/6/G1091    most recent
00049.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (8)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Nonome, K.
Right arrow Articles by Sugiyama, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Nonome, K.
Right arrow Articles by Sugiyama, T.

LIVER AND BILIARY TRACT

Human umbilical cord blood-derived cells differentiate into hepatocyte-like cells in the Fas-mediated liver injury model

Kazunobu Nonome,1,2 Xiao-Kang Li,1 Terumi Takahara,2 Yusuke Kitazawa,1 Naoko Funeshima,1 Yutaka Yata,2 Feng Xue,2 Masami Kanayama,2 Eiji Shinno,2 Chieko Kuwae,3 Shigeru Saito,4 Akiharu Watanabe,2 and Toshiro Sugiyama2

1Laboratory of Transplantation Immunology, National Research Institute for Child Health and Development, Tokyo; 2Third Department of Internal Medicine, Toyama Medical and Pharmaceutical University, Toyama; 3Department of Obstetrics and Gynecology, Tokyo Metropolitan Fuchu Hospital, Tokyo; and 4Department of Obstetrics and Gynecology, Toyama Medical and Pharmaceutical University, Toyama, Japan

Submitted 8 February 2005 ; accepted in final form 21 June 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Human umbilical cord blood (HUCB) contains stem/progenitor cells, which can differentiate into a variety of cell types. In this study, we investigated whether HUCB cells differentiate into hepatocytes in vitro and in vivo. We also examined whether CD34 could be the selection marker of stem cells for hepatocytes. HUCB cells were obtained from normal full-term deliveries, and CD34+/– cells were further separated. For in vitro study, HUCB cells were cultured for 4 wk, and expressions of liver-specific genes were examined. For the in vivo study, nonobese diabetic/severe combined immunodeficient mice were subjected to liver injury by a Fas ligand-carried adenoviral vector or only radiated. Mice were treated simultaneously with or without cell transplantation of HUCB, CD34+, or CD34– cells. After 4 wk, human-specific gene/protein expression was examined. In the in vitro study, human liver-specific genes were positive after 7 days of culture. The immunofluorescent study showed positive staining of {alpha}-fetoprotein, cytokeratin 19, and albumin in round-shaped cells. In the in vivo study, immunohistochemical analysis showed human albumin-positive, hepatocyte-specific antigen-positive cells in mouse livers of the Fas ligand/transplantation group. Fluorescence in situ hybridization analysis using the human Y chromosome also showed positive signals. However, no difference between transplanted cell types was detected. In contrast, immunopositive cells were not detected in the irradiated/transplantation group. The RT-PCR result also showed human hepatocyte-specific gene expressions only in the Fas ligand/transplantation group. HUCB cells differentiated into hepatocyte-like cells in the mouse liver, and liver injury was essential during this process. The differences between CD34+ and CD34– cells were not observed in human hepatocyte-specific expression.

cell transplantation; Fas ligand; human umbilical cord blood; hepatocyte; liver injury

STEM CELLS are defined as undifferentiated long-lived cells that are capable of multiple rounds of division. Human umbilical cord blood (HUCB) is reported to contain stem/progenitor cells in greater numbers than the bone marrow or adult peripheral blood (4, 14, 39). Recently, successful HUCB cell transplantation for various blood diseases has resulted in a lower incidence of graft versus host disease than conventional therapy (32, 44). In these clinical applications, hematopoietic stem cells can differentiate into mature blood cells. CD34+ cells, which carry the well-known hematopoietic stem cell marker, have the capacity to differentiate into all blood cell lineages (7, 14, 35). HUCB cells have also been reported to differentiate into a variety of cell types such as bone (9, 13), cartilage (9), muscle (9) and nerve cells (5, 13) and hepatocytes (15, 17, 27, 40).

Recent studies have shown that male recipients of female liver allografts have a small number of male hepatocytes within the graft in long-term observations (20, 33). Other recent studies also have shown that bone marrow cells could differentiate into hepatocytes in the damaged liver (1, 3, 6, 21, 30). These results suggested that the bone marrow contains a certain number of hepatic progenitor cells (3, 30). Oval cells in the canal of Herring may proliferate after hepatic injury, when the growth of mature hepatocytes is suppressed (34, 37). In addition, oval cells can differentiate into cholangiocytes in response to various types of stress or liver injury. From all these studies, it remains unclear which cell type actually differentiates into the hepatocyte during liver injury. Although hepatic progenitor cells exist in the bone marrow and HUCB, it is still unclear whether these cells differentiate into hepatocytes or fuse with host hepatocytes (2, 15, 25, 36, 41, 42). Recent studies have shown that oval cells express CD34, Thy-1, and c-Kit, all of which are also found in hematopoietic stem cells (30). For this reason, we postulated that CD34+ cells in HUCB could differentiate into hepatocytes in the hepatocyte-specific niche.

From clinical application to cell therapy, there are many problems with bone marrow cell transplantation, such as the limitation of cell number, the donor’s physical responsibility, and the prevention of rejection. HUCB, however, can be collected without any harm to the newborn infant and has been found to be multipotent and cause fewer episodes of rejection because of the immaturity. Therefore, the applications of HUCB cells should be further expanded and may serve as an excellent alternative to bone marrow-derived stem cells for cell transplantation in various diseases. In this study, we investigated whether HUCB cells differentiate into hepatocytes in vivo and whether CD34 can be the selection marker of stem cells for the hepatocyte.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
HUCB Cells

Samples of male HUCB were obtained during normal full-term deliveries with informed consent, which was approved by the institutional Ethics Review Committee. HUCB was centrifuged at 1,500 rpm for 35 min at 25°C on a layer of Ficoll-Paque (Amersham-Pharmacia Biotech; Little Chalfont, UK). The mononuclear cell layer was suctioned, followed by a reaction with fluorescence-activated cell sorting (FACS) lysing solution (Becton Dickinson; San Jose, CA). After being washed with PBS, cells were first dissolved with PBS, FcR, and CD34 MicroBeads (Miltenvi Biotec; Auburn, CA), according to the manufacturer’s instructions. The cells were incubated for 20 min at 4°C and washed with PBS. The isolation of CD34+ cells was performed using Auto MACS (Miltenvi Biotech). Isolated CD34+ cells were stained with both CD34-PE antibody and lineage cocktail-FITC antibody (Becton Dickinson) and analyzed with FACS (Becton Dickinson). All the following studies were performed when the purity of CD34+ and lineage cocktail– cells was >80%.

Cell Culture

HUCB cells and CD34+ or CD34– cells were cultured in DMEM supplemented with 15% FBS, 100 U/ml penicillin, 100 U/ml streptomycin, and growth factors including hepatocyte growth factor (20 ng/ml) and fibroblast growth factor (10 ng/ml). HUCB cells were plated on collagen-coated dishes and maintained at 37°C in a 5% CO2 atmosphere. Medium change was carried out twice weekly thereafter.

Animal Model

Eight- to ten-week-old nonobese diabetic (NOD)/severe combined immunodeficient (SCID) female mice purchased from Clea Japan (Tokyo, Japan) were used. All mice were maintained under specific pathogen-free conditions in the animal center with free access to water and food. Animal care was in accordance with the guidelines of the National Research Institute for Child Health and Development. The mice were first divided into two groups (liver injury group and irradiation group). The liver injury group was injected with an adenoviral vector encoding Fas ligand (FasL) (AxCALNFasL) and Cre (AxCALNCre) via the tail vein (10, 28). The mice were simultaneously treated with cell transplantation (FasL/TP), and mice without transplantation were used as a control (FasL). The irradiated group was irradiated 250 cGy before the cell transplantation (Ra/TP). Cell transplantation was performed with 5x104 mononuclear cells, either CD34+ or CD34– cells. The recipient mice were intraperitoneally injected with 20 µl of anti-asialo GM1 antibody (Wako; Osaka, Japan) to delete the natural killer cells before the cell transplantation. Subsequent antibody treatments were performed on days 11 and 22. Four weeks after the cell transplantation, animals were killed, and peripheral blood, liver tissues, the spleen, the tibia, and the femur were collected.

Measurement of Alanine Aminotransferase

Alanine aminotransferase (ALT) was assayed by test paper using the Reflotron checker (Reflotron, Roche; Basel, Switzerland). A normal value for ALT was <40 U/l.

FACS Analysis

Mouse peripheral blood samples were centrifuged at 3,000 rpm for 20 min at 25°C on a layer of Ficoll-Paque. Collected mononuclear cells were incubated with 20 µl human CD45-PE antibody (Becton Dickinson) and processed for FACS analysis.

Bone marrow cells were collected by tibial and femural aspiration with PBS using a syringe. Cell suspensions were then filtered through a cell strainer and processed for FACS analysis. The spleen was rubbed with slides to liberate cells. Cell suspensions were then filtered through a cell strainer and analyzed by FACS.

In addition, purified HUCB cells were incubated with human anti-Fas antibody (Becton Dickinson) to examine Fas expression on HUCB cells and processed for FACS analysis. Human peripheral blood from healthy volunteers was used as a positive control.

RT-PCR

Total RNA was extracted from frozen mouse liver tissue and cultured cells using ISOGEN (Nippon Gene; Tokyo, Japan), according to the manufacturer’s instructions. Normal human liver tissue obtained from normal areas during the surgical resection of hepatocellular carcinoma was used as a control. RNA was purified to remove contaminating DNA according to the DNA-free protocol (Ambion; Austin, TX) and quantified using a spectrophotometer. Equal amounts of RNA were subjected to cDNA synthesis using oligo dT (Invitrogen; Carlsbad, CA). The obtained cDNA was amplified with Zymoreactor (ATTO; Tokyo, Japan) according to the conditions described in Table 1. The sequences and product sizes for human albumin, {alpha}-fetoprotein ({alpha}-FP), glutamine synthetase (GS), transferrin, cytokeratin 19 (CK-19), {beta}-actin, and mouse GAPDH are shown in Table 1. The amplified products were electrophoresed on 1.5% agarose gels and stained with ethidium bromide.


View this table:
[in this window]
[in a new window]
  		Table 1. RT-PCR primers and conditions for each annealing temperature, cycle number of amplification, and product size

 
Immunofluorescent Staining Analysis of Cultured Cells

Cultured HUCB cells were fixed with acetone for 10 min. After being washed with PBS, cells were incubated with primary antibodies at room temperature for 1 h. For primary antibodies, anti-human albumin-FITC (DAKO; Kyoto, Japan), CK-19 (DAKO), and {alpha}-FP (DAKO) antibodies were used. The slides were then washed with PBS and incubated for 30 min at room temperature with anti-mouse IgG TRITC conjugate (Sigma) or phycoprobe R-PE anti-goat IgG (Biomeda). After being washed, slides were immediately applied with 4,6-diamidino-2-phenylindole (DAPI, Vector Laboratories) and then analyzed by fluorescent microscope (model AX80T, Olympus; Tokyo, Japan).

To evaluate the number of immunostained cells, photographs of 10 random fields (x100) per slide were analyzed by NIH Image software (National Institutes of Health; Bethesda, MD).

Immunohistochemistry

Horseradish peroxidase-conjugated sheep anti-human albumin antibody (Serotec; Oxford, UK) was used to avoid a nonspecific reaction with the mouse liver as follows: NOD/SCID mice were injected with antibody via tail vein, and peripheral blood was collected in 15 min. After centrifugation, the serum was inactivated at 56°C for 30 min.

Mononuclear cells collected from HUCB were transferred to slides by the cytospin method. The slides of 5-µm frozen liver sections were fixed with acetone followed by formol calcium for 2 min. Endogenous peroxidase was blocked for 10 min using 0.3% H2O2 in PBS. After being washed with PBS, sections were incubated with purified human albumin antibody for 3 h at room temperature. The staining was visualized with diaminobenzidine (DAB). Anti-human hepatocyte antibody (DAKO), which specifically recognizes human hepatocytes, was used as the primary antibody. Immunostaining was visualized using a biotin-conjugated secondary antibody according to the manufacturer’s instruction. To evaluate the number of immunostained cells and the immunopositive area, photographs of five random fields (x100) per slide were analyzed by NIH Image software.

Fluorescence In Situ Hybridization

Human Y chromosome DNA probe CEP Y (Vysis) was used. The slides of 5-µm frozen liver sections were fixed with methanol for 30 min. After being washed with PBS, slides were incubated with 2x SSC-0.1% Nonidet P-40 (Nacalai Tesque; Kyoto, Japan) for 30 min at 37°C and dehydrated with ethanol. The sections were denatured with 70% formamide-2x SSC at 73°C for 5 min and then hybridized with the denatured probe overnight at 37°C. After being washed, slides were immediately applied with DAPI and then analyzed by fluorescent microscope (model AX80T).

Statistical Analysis

Results are expressed as means ± SD, and a statistical analysis was performed with Student’s t-test. P < 0.05 was considered as a significant difference between groups. A statistical evaluation for graft survival was performed using the Kaplan-Meier test. P values <0.05 were considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
In Vitro Studies

Cultured HUCB cells. HUCB cells were differentiated into both round and spindle-shaped cells by day 7 (Fig. 1A). The cytoplasm of the round cells was enlarged, whereas the spindle-shaped cells had differentiated into fibroblast-like cells during the time course (Fig. 1B). However, when CD34+ or CD34– cells were cultured, living cells were decreased gradually during the time course. The rate of round cells in all cultured cells was about 70%, which did not change during incubation (Fig. 1C).



View larger version (93K):
[in this window]
[in a new window]
  		Fig. 1. Cultured human umbilical cord blood (HUCB) cells. A: cultured HUCB cells were differentiated into round and spindle-shaped cells morphologically. B: whereas the cytoplasm of the round cells was enlarged, the spindle-shaped cells had differentiated into fibroblast-like cells during the time course. C: the rate of the round cell number in all cultured cells did not change.

 
Immunofluorescent staining analysis. The immunofluorescent study showed positive expression of human {alpha}-FP, albumin, and CK-19 in the round cells but not in the fibroblast-like cells (Fig. 2). {alpha}-FP-positive staining was observed with a peak on day 7 that then decreased gradually until day 28. In contrast, the rate of albumin- and CK-19-positive cells increased during the longer culture. Albumin-positive cells accounted for about 50% of all cultured cells on days 21 and 28 (Fig. 3). Cultured CD34+ and CD34– cells were also positive for human {alpha}-FP, albumin, and CK-19, although the numbers of living cells decreased during incubation (data not shown).



View larger version (49K):
[in this window]
[in a new window]
  		Fig. 2. Immunofluorescent staining of cultured HUCB cells. A: cells (day 7) were stained with human {alpha}-fetoprotein [{alpha}-FP (red)]. B: cells (day 14) were stained with both human albumin (green) and {alpha}-FP (red) (arrow). C: cells (day 21) were stained with human albumin (green). D: cells (day 14) were stained with both human albumin (green) and cytokeratin 19 [CK-19 (red)] (arrows). E: cells (day 21) were stained with human CK-19 (red). Nuclei were stained with DAPI (blue).

 


View larger version (11K):
[in this window]
[in a new window]
  		Fig. 3. Expression rate of immunofluorescent staining in cultured HUCB cells. {alpha}-FP-positive cells were peaked on day 7 and then decreased gradually until day 28. The rate of albumin- and CK-19-positive cells in all cultured cells increased during the longer culture. Albumin-positive cells accounted for about 50% at days 21 and 28.

 
RT-PCR assay. RT-PCR was performed to detect human-specific expression of albumin, {alpha}-FP, GS, transferrin, and CK-19 (Fig. 4). The normal human liver was used as a positive control. Freshly isolated HUCB cells did not express these genes. However, HUCB cells expressed all these genes by day 14.



View larger version (41K):
[in this window]
[in a new window]
  		Fig. 4. Expression of human-specific genes in cultured HUCB cells by RT-PCR. Lane A, normal human liver; lane B, HUCB cells on day 0; lane C, HUCB cells on day 7; lane D, HUCB cells on day 14; lane E, HUCB cells on day 21; lane F, HUCB cells on day 28. {beta}-Actin was used as an internal control. GS, glutamine synthase; AFP, {alpha}-FP.

 
In Vivo Studies

Low-level Fas expression in HUCB cells. We first examined the expression of Fas antigen on the surface of HUCB cells. As shown in Fig. 5, very low expression of Fas antigen on HUCB cells compared with human peripheral blood cells, which strongly express Fas, was observed. These results indicated that the transplanted HUCB cells would not be damaged by the Fas-mediated liver injury model used in this study.



View larger version (12K):
[in this window]
[in a new window]
  		Fig. 5. Fluorescence-activated cell sorting analysis showed the expression of Fas antigen in human peripheral blood cells and HUCB cells. A: human peripheral blood cells strongly expressed Fas antigen (98.5%). B: HUCB cell expression of Fas antigen was very low (4.5%).

 
Fas-mediated liver injury. To evaluate the amount of recombinant adenovirus vector AxCALNFasL administration, mice were administered 4.2 x 108, 4.8 x 108, or 5.3 x 108 adenoviral plaque-forming units (pfu), respectively. The amount of recombinant adenoviral vector AxCALNCre was 1.0 x 109 pfu. The peak ALT level after three doses was on day 3, and the values were 480.8 ± 120.8 IU/l (Fig. 6). Hepatic apoptosis and necrosis were observed in all groups as detected by hematoxylin-eosin (HE) staining, although the degree was almost the same in each group. Vascular architecture was intact in all groups. The liver damage was transient, and histology was nearly normal on day 10 in the surviving mice (Fig. 6C). Because the hepatic necrosis areas shown by the HE stain were equal, mice were administered 4.8 x 108 pfu AxCALNFasL to induce liver injury.



View larger version (92K):
[in this window]
[in a new window]
  		Fig. 6. Histology of Fas-mediated liver injury. A: hematoxylin-eosin staining of the mouse liver on day 3 after treatment. Hepatic apoptosis and hemorrhagic necrosis were widely observed in the liver. Vascular architecture was intact. B: mouse liver on day 10 after treatment. The liver injury was transient, and the histology had recovered to normal by day 10. C: alanine aminotransferase (ALT) concentrations by liver injury induced by Fas ligand (FasL) recombinant adenoviral vector treatment. Mice were administered 4.2 x 108 (a), 4.8 x 108 (b), or 5.3 x 108 plaque-forming units (c) of adenovirus, respectively. The peak of ALT concentration was significantly higher on day 3 in all doses after the treatment, and the values were 476.5 ± 160, 478 ± 65.1, and 418.7 ± 46.8 IU/l, respectively (n = 5). P < 0.05 vs. day 0.

 
FACS analysis. Mice were killed 1 mo after HUCB cell transplantation, and the population of HUCB-derived leukocytes was studied using anti-human CD45 antibody on the spleen, bone marrow, and peripheral blood in both the FasL/TP and Ra/TP groups by FACS analysis (Fig. 7). In the FasL/TP group, CD45+ leukocytes were observed only in the peripheral blood. In contrast, CD45+ cells were detected in the spleen, bone marrow, and peripheral blood in the Ra/TP group.



View larger version (23K):
[in this window]
[in a new window]
  		Fig. 7. Human CD45 expression was studied in spleen, the bone marrow, and peripheral blood cells 1 mo after cell transplantation. A: FasL/transplantation (FasL/TP) group. CD45+ cells were observed only in peripheral blood in the FasL/TP group, and the value was 59.1%. Bone marrow cells without a reaction to the antibody were used as a negative control. B: radiation (Ra)/TP group. CD45+ cells were detected in the spleen, bone marrow, and peripheral blood cells in the Ra/TP group, and the value was 33.8%, 14.1%, and 14.7%, respectively.

 
Immunohistochemistry. Mononuclear HUCB cells were transferred to the slide by cytospin, stained with human albumin antibody, and confirmed to be negative (Fig. 8A). The NOD/SCID mouse liver was also negative for human albumin (Fig. 8B). In the Ra/TP group, human albumin-positive cells were not observed, although three different sections in each mouse were examined (n = 15; data not shown). In contrast, in the FasL/TP group, human albumin-positive cells were easily detected (Fig. 8, C and D), as follows: 0.39% (0 ~ 0.98, n = 5), 0.40% (0 ~ 0.73, n = 4), and 0.39% (0 ~ 0.68, n = 5) in the transplanted group with CD34+, CD34–, and mononuclear cells, respectively. The area of reactivity was as follows: 0.24% (0 ~ 0.76, n = 5), 0.21% (0 ~ 0.42, n = 4), and 0.11% (0 ~ 0.24, n = 5) in the transplanted group with CD34+, CD34–, and mononuclear cells, respectively. The difference, however, was not statistically significant (Fig. 9). The cells that reacted to the anti-albumin antibody were sparsely detected. These cells were located in the parenchyma and the circumference of vessels in each group. Clusters of positive cells were detected, although very rarely (date not shown). In a long-term study, the positive cells were still observed 5 mo after cell transplantation, and the rate decreased during the time course (data not shown).



View larger version (70K):
[in this window]
[in a new window]
  		Fig. 8. Immunohistochemical studies of human albumin and hepatocyte-specific antigen staining. A: cytospin of HUCB cells demonstrated mononuclear cells of HUCB stained with anti-human albumin antibody. B: in the nonobese diabetic (NOD)/severe immunodeficient (SCID) mouse liver of the FasL group, the hepatocytes did not express human albumin. C: CD34+ cell transplantation. D: CD34– cell transplantation. E: CD34+ cell transplantation. F: mononuclear cell transplantation. G: CD34– cell transplantation. H: CD34+ cell transplantation. In the FasL/TP group, human albumin-positive cells were detected, and the cell sizes and shapes were similar to those of mature hepatocytes. The expression was similar in all three groups. Cells expressing human albumin existed in the parenchyma (C and D). Immunostaining of human hepatocyte-specific antigen was positive in the FasL/TP group (E and F). The FasL and Ra/TP groups did not express human albumin and hepatocyte-specific antigen. G and H: fluorescence in situ hybridzatin (FISH) for human Y chromosome. Cells expressing human Y chromosome existed in the parenchyma (G) and the circumference of vessel (V; H).

 


View larger version (11K):
[in this window]
[in a new window]
  		Fig. 9. Expression rates (A) and areas (B) in the FasL/TP group. A: the expression rate was as follows: 0.39% (0 ~ 0.98, n = 5), 0.40% (0 ~ 0.73, n = 4), and 0.39% (0 ~ 0.68, n = 5) in FasL/TP group with CD34+, CD34–, or HUCB cells, respectively. B: the expression area was as follows: 0.24% (0 ~ 0.76, n = 5), 0.21% (0 ~ 0.42, n = 4), and 0.11% (0 ~ 0.24, n = 5) in the FasL/TP group with CD34+, CD34–, or HUCB cells, respectively. ND, not statistically different.

 
Immunostaining of human hepatocyte-specific antigen was also positive in the FasL/TP group, and reactivity against anti-human hepatocytes was similar in all groups (Fig. 8, E and F). FasL and Ra/TP groups were negative (data not shown).

FISH. FISH for human Y chromosome showed positive signals on sections of the FasL/TP group (Fig. 8, G and H). However, FasL and Ra/TP groups were negative (data not shown). The positive cells existed in the parenchyma (Fig. 8G) and in the circumference of vessels (Fig. 8H) in the FasL/TP group.

RT-PCR assay. RT-PCR was performed to detect human-specific expression of albumin, {alpha}-FP, GS, transferrin, and CK-19 (Fig. 10). The normal human liver was used as a positive control. Freshly isolated HUCB cells and the mouse liver of the FasL group did not express these genes. The human liver demonstrated these expressions. In the FasL/TP group, these genes were all expressed except for CK-19, and the expression was not different in each transplantation group of CD34+, CD34–, and mononuclear cells. These results suggested that transplanted cells differentiated into hepatocyte-like cells, and these three groups showed similar results.



View larger version (56K):
[in this window]
[in a new window]
  		Fig. 10. Expression of human-specific genes of albumin, {alpha}-FP, transferrin, and GS by RT-PCR in the mouse liver. Lane A, normal human liver; lane B, HUCB cells; lane C, FasL group; lane D, CD34+ cell transplantation of the FasL/TP group; lane E, CD34– cell transplantation of the FasL/TP group; lane F, HUCB cell transplantation of the FasL/TP group; lane G, Ra/TP group. Human {beta}-actin and mouse (m) GAPDH genes were used as internal controls.

 
Survival rate. In our experiment, some mice of the FasL group died within 6 days, and the final survival rate was 70% (Fig. 11). However, none of the FasL/TP group mice died, and the rate was significantly higher, with a final result of 100% (P < 0.05).



View larger version (8K):
[in this window]
[in a new window]
  		Fig. 11. The survival rate of FasL group mice was 70%, whereas that of FasL/TP group mice was 100%.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the present study, we showed cultured CD34+, CD34–, and HUCB cells could differentiate into the cells expressing human liver-specific genes and proteins by RT-PCR assay and immunofluorescent staining. In addition, we showed that transplanted CD34+, CD34–, and HUCB cells expressed human hepatocyte-specific genes and proteins by an RT-PCR assay and immunohistochemical study in the injured mouse liver, respectively. FISH analysis also confirmed the presence of human Y chromosome in the female mouse liver. HUCB cells and mouse hepatocytes did not express these human genes and proteins; therefore, the transplanted CD34+, CD34–, and HUCB cells differentiated into hepatocyte-like cells in this Fas-mediated liver injury model. In addition, the hepatocyte-like cells may be hepatic progenitor cells or mature hepatocyte-like cells, because {alpha}-FP is known to be expressed in hepatic progenitor cells, including oval cells (10, 31), and GS is a marker of mature hepatocytes (12, 26). Previous reports of HUCB cells have shown that albumin-producing cells have been detected at 21 days in a primary culture system containing growth and differentiation factors (17) and at 4 wk after transplantation in vivo (17), respectively, which is consistent with our study.

Wagers et al. (38) showed that single transplanted hematopoietic stem cells differentiated at a very low level of into hepatocytes in mice without liver dysfunction and that transplanted cells did not express albumin. The present study has also shown that transplanted HUCB cells could differentiate into hepatocyte-like cells only, but not cholangiocytes, in the Fas-mediated injured liver. Because cultured HUCB cells could differentiate into cholangiocyte-like cells, which express CK-19, this might be the reason that this model did not damage bile ducts. Liver injury and the surrounding environment may be essential for transplanted cells to be engrafted in the liver and to differentiate into hepatocytes (15, 17, 27, 38, 40). Recent studies have suggested that stromal cell-derived factor and its receptor CXCR4 might be important for stem cell recruitment to the liver (18, 29). In addition, CK-19, which is a differentiation marker of liver progenitor cells into bile duct cells, was not detectable in this study. This finding may explain why the bile ducts were not damaged in our liver injury model.

Recent studies have further shown that transplanted HUCB cells could differentiate into cells that express human albumin in carbon tetrachloride-induced (17) or 2-acetylaminofluorene-induced (15) liver injury models. Our animal model induced rapid and massive apoptosis in the liver by the FasL recombinant adenoviral expresson vector AxCALNFasL. FasL expression induces hepatocyte apoptosis both by a direct interaction with Fas in hepatocytes and by Fas-positive inflammatory cells (28). The primary cause of liver damage used in this study is the Fas/FasL pathway, and this liver injury may be similar to human viral hepatitis rather than drug-induced injury, such as that by carbon tetrachloride or 2-acetylaminofluorene. In that context, the present study demonstrates clinical applications worth considering.

We observed that the Ra/TP group expressed human CD45+ leukocytes not only in peripheral blood but also in the spleen and bone marrow, which confirmed that the transplantation was successful, because CD34+ cells differentiate into blood cells (14, 35, 44). The FasL/TP group expressed CD45+ cells only in peripheral blood. The mechanism of this phenomenon is that transplanted cells enter the bone marrow and differentiate into blood cells but do not survive long term and are excluded because the bone marrow is not damaged by radiation treatment in this liver injury model.

Although we postulated that HUCB-derived CD34 (a hematopoietic stem cell marker)-expressing cells may be an important marker for hepatocyte differentiation, a difference was not observed between the CD34+ and CD34– cells that differentiated into human hepatocyte-specific expression cells in our study. Even though the number of selected cells was not high, at 100% human albumin-positive cells did not increase in number in several experiments using 98% purified cells, which suggested that CD34+ or CD– cells exhibit no difference in terms of differentiation to hepatocytes.

Wang et al. (40) showed that both CD34+ and CD34+-CD38+-CD7– cells from HUCB and bone marrow differentiat into hepatocyte-like cells by liver injury. Normal adult bone marrow was also reported to contain a small number of hepatic progenitor cells, which can differentiate into hepatocytes when the growth of mature hepatocytes is suppressed by hepatic injury (1, 3, 6, 21). Although it is still unclear which cell types of bone marrow and HUCB cells differentiate into hepatocytes, CD34 (26, 30), CD38 (40), c-Kit (11, 30), Thy-1 (11, 30, 31), and Liv-8 (43) may be candidates. However, differentiation into hepatocytes may be multifactorial. Further study is needed to determine the cell marker for liver cell differentiation. However, Lee et al. (22) showed that human mesenchymal stem cells derived from bone marrow and cord blood were effectively differentiated into hepatocyte-like cells in vitro. Because stem cells have plasticity, cells may differentiate into hepatocytes under conditions of hepatic injury without selecting HUCB by cell markers.

Recent studies have suggested that bone marrow cells are pluripotent and are able to become mature hepatocytes. Several groups have attributed this apparent plasticity to transdifferentiation. Willenbring et al. and others (2, 25, 36, 41, 42), however, showed that bone marrow-derived hepatocytes emerged from fusion between donor bone marrow-derived cells and host hepatocytes, and they suggested that cell fusion could explain these results. They also suggested that hepatocytes derived from HUCB cell transplantation might arise from cell fusion. Newsome et al. (27), however, showed that HUCB cells could differentiate into hepatocytes in the absence of cell fusion. We have not investigated whether hepatocytes derived from HUCB cells arise from cell fusion. Further study is necessary to evaluate the role of cell fusion in our study.

In our experiment, the survival rate of FasL group mice was 70%, whereas that of FasL/TP group mice was 100% (P < 0.05). FasL expression induced hepatocyte apoptosis by a direct interaction with Fas of hepatocytes and by Fas-positive inflammatory cells, which caused the death of the mice in the FasL group within 6 days. Transplanted cells may play a role in the survival of the mice in early-phase Fas-mediated liver injury. One possibility is that transplanted cells secrete some anti-inflammatory factors that modify the immunological state so as to improve the survival rate (8, 19, 24). The other possibility is that transplanted HUCB cells may heal liver injury in our model, although only a small number of albumin-expressing cells were observed in our study. A recent study (16) in which hematopoietic stem cells were transplanted into CCl4 injured mice demonstrated that 7.6% of the total liver cells were human albumin-positive cells, and serum ALT, prothrombin time, and fibrinogen recovered as early as 2 days after transplantation; however, the precise mechanism has not been clarified. Similar to this report, it is possible that HUCB cell transplantation can suppress liver injury in our previous model. However, the reasons why the survival rate improved in Fas-mediated liver injury mice is still unclear, and further study is needed to elucidate this question.

In summary, the present study shows that CD34+, CD34–, and HUCB cells differentiate into hepatocyte-like cells in this Fas-mediated liver injury model and that the survival rate of transplanted mice was improved.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported in part by Ministry of Education, Culture, Sports, Science and Technology of Japan Grant-In-Aid 14657293 and a grant for the Organized Research Combination System.


   ACKNOWLEDGMENTS
 
The authors are grateful to Dr. S. Enosawa for useful suggestions and comments. They also thank M. Wakabayashi and N. Takahashi for the technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: X.-K. Li, Laboratory of Transplantation Immunology, National Research Institute for Child Health and Development, 2-10-1 Okura, Setagaya-ku, Tokyo 157-8535, Japan (e-mail: sri{at}nch.go.jp)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Alison MR, Poulsom R, Jeffery R, Dhillon AP, Quaglia A, Jacob J, Novelli M, Prentice G, Williamson J, and Wright NA. Hepatocytes from non-hepatic adult stem cells. Nature 406: 257, 2000.[CrossRef][Medline]
  2. Alvuares-Dolado M, Pardal R, Garcia-Verdugo JM, Fike JR, Lee HO, Pfeffer K, Lois C, Morrison SJ, and Alvarez-Buylla A. Fusion of bone-marrow-derived cells with Purkinje neuron, cardiomyocytes and hepatocytes. Nature 425: 968–973, 2003.[CrossRef][Medline]
  3. Avital I, Inderbitzin D, Aoki T, Tyan DB, Cohen AH, Ferraresso C, Rozga J, Arnaout WS, and Demetriou AA. Isolation, characterization, and transplantation of bone marrow-derived hepatocyte stem cells. Biochem Biophys Res Commun 288: 156–164, 2001.[CrossRef][Web of Science][Medline]
  4. Broxmeyer HE, Douglas GW, Hangoc G, Cooper S, Bard J, English D, Arny M, Thomas L, and Boyse EA. Human umbilical cord blood as a potential source of transplantable hematopoietic stem/progenitor cells. Proc Natl Acad Sci USA 86: 3828–3832, 1989.[Abstract/Free Full Text]
  5. Buzanska L, Machaj EK, Zablocka B, Pojda Z, and Domanska-Janik K. Human cord blood-derived cells attain neuronal and glial features in vitro. J Cell Sci 115: 2131–2138, 2002.[Abstract/Free Full Text]
  6. Dahlke MH, Popp FC, Bahlmann FH, Aselmann H, Jäger MD, Neipp M, Piso P, Klempnauer J, and Schlitt HJ. Liver regeneration in a retrosine/CCl4-induced acute liver failure model: do bone marrow-derived cells contribute? J Hepatol 39: 365–373, 2003.[CrossRef][Web of Science][Medline]
  7. DiGiusto DL, Lee R, Moon J, Moon J, Moss K, O’Toole T, Voytovich A, Webster D, and Mule JJ. Hematopoietic potential of cryopreserved and ex vivo manipulated umbilical cord blood progenitor cells evaluated in vitro and in vivo. Blood 87: 1261–1271, 1996.[Abstract/Free Full Text]
  8. Dinarello CA. Interleukin-1 and interleukin-1 antagonism. Blood 77: 1627–1652, 1991.[Abstract/Free Full Text]
  9. Erices A, Conget P, and Minguell JJ. Mesenchymal progenitor cells in human umbilical cord blood. Br J Haematol 109: 235–242, 2000.[CrossRef][Web of Science][Medline]
  10. Evarts RP, Nagy P, Nakatsukasa H, Marsden E, and Thorgeirsson SS. In vivo differentiation of rat liver oval cells into hepatocytes. Cancer Res 49: 1541–1547, 1989.[Abstract/Free Full Text]
  11. Fiegel HC, Lioznov MV, Cortes-Dericks L, Lange C, Kluth D, Fehse B, and Zander AR. Liver-specific gene expression in cultured human hematopoietic stem cells. Stem Cells 21: 98–104, 2003.[Abstract/Free Full Text]
  12. Gaasbeek Janzen JW, Gebhardt R, Ten Voorde GHJ, Lamers WH, Charles R, and Moorman AFM. Heterogenous distribution of glutamine synthetase during rat liver development. J Histochem Cytochem 35: 49–54, 1987.[Abstract]
  13. Goodwin HS, Bickness AR, Chien SN, Bogucki BD, Quinn CO, and Wall DA. Multilineage differentiation activity by cells isolated from umbilical cord blood; expression of bone, fat, and neural markers. Biol Blood Marrow Transplant 7: 581–588, 2001.[CrossRef][Web of Science][Medline]
  14. Hao QL, Shah AJ, Thiemann FT, Smogorzewska EM, and Crooks GM. A functional comparison of CD34+ CD38– cells in cord blood and bone marrow. Blood 86: 3745–3753, 1995.[Abstract/Free Full Text]
  15. Ishikawa F, Drake CJ, Yang S, Fleming PA, Minamiguchi H, Visconti RP, Crosby CV, Argraves WS, Harada M, Key LL, Livingston AG, Wingard JR, and Ogawa M. Transplanted human cord blood cells give rise to hepatocytes in engrafted mice. Ann NY Acad Sci 996: 174–185, 2003.[CrossRef][Web of Science][Medline]
  16. Jang YY, Collector MI, Baylin SB, Diehl AM, Sharkis SJ. Hematopoietic stem cells convert into liver cells within days without fusion. Nat Cell Biol 6: 532–539, 2004.[CrossRef][Web of Science][Medline]
  17. Kakinuma S, Tanaka Y, Chinzei R, Watanabe M, Shimizu-Saito K, Hara Y, Teramoto K, Arii S, Sato C, Takase K, Yasumizu T, and Teraoka H. Human umbilical cord blood as a source of transplantable hepatic progenitor cells. Stem Cells 21: 217–227, 2003.[Abstract/Free Full Text]
  18. Kollet O, Shivtiel S, Chen YQ, Suriawinata J, Thung SN, Dabeva MD, Kahn J, Spiegel A, Samira S, Goichberg P, Kalinkovich A, Arenzana-Seisdedos F, Nagler A, Hardan I, Revel M, Shafritz DA, and Lapidot T. HGF, SDF-1, and MMP-9 are involved in stress-induced human CD34+ stem cell recruitment to the liver. J Clin Invest 112: 160–169 2003.[CrossRef][Web of Science][Medline]
  19. Kopf M, Gros GL, Bachmann M, Lamers MC, Bluethmann H, and Köhler G. Disruption of the murine IL-4 gene blocks Th2 cytokine responses. Nature 362: 245–248, 1993.[CrossRef][Medline]
  20. Körbling M, Katz RL, Khanna A, Ruifrok AC, Rondon G, Albitar M, Champlin RE, and Estrov Z. Hepatocytes and epithelial cells of donor origin in recipients of peripheral-blood stem cells. N Engl J Med 346: 738–746, 2002.[Abstract/Free Full Text]
  21. Lagasse E, Connors H, Al-Dhalimy M, Reitsma M, Dohse M, Osborne L, Wang X, Finegold M, Weissman IL, and Grompe M. Purified hematopoietic stem cells can differentiate into hepatocytes in vivo. Nat Med 6: 1229–1234, 2000.[CrossRef][Web of Science][Medline]
  22. Lee KD, Kuo TKC, Whang-Peng J, Chung YF, Lin CT, Chou SH, Chen JR, Chen YP, and Lee KS. In vitro hepatic differentiation of human mesenchymal stem cells. Hepatology 40: 1275–1284, 2004.[CrossRef][Web of Science][Medline]
  23. Li XK, Fujino M, Sugioka A, Morita M, Okuyama T, Guo L, Funeshima N, Kimura H, Enosawa S, Amemiya H, and Suzuki S. Fulminant hepatitis by Fas-ligand expression in MRL-lpr/lpr mice grafted with Fas-positive livers and wild-type mice with Fas-mutant livers. Transplantation 71: 503–508, 2001.[CrossRef][Web of Science][Medline]
  24. Marino MW, Dunn A, Grail D, Inglese M, Noguchi Y, Richards E, Jungbluth A, Wada H, Moore M, Williamson B, Basu S, and Old LJ. Characterization of tumor necrosis factor-deficient mice. Proc Natl Acad Sci USA 94: 8093–8098, 1997.[Abstract/Free Full Text]
  25. Medvinsky A and Smith A. Fusion brings down barriers. Nature 422: 823–825, 2003.[CrossRef][Medline]
  26. Moorman AF, Vermeulen JLM, Charles R, and Lamers WH. Localization of ammonia-metabolizing enzymes in human liver. Hepatology 9: 367–372, 1989.[Web of Science][Medline]
  27. Newsome PN, Johannessen I, Boyle S, Dalakas E, McAulay KA, Samuel K, Rae F, Forrester L, Turner ML, Hayes PC, Harrison DJ, Bickmore WA, and Plevris JN. Human cord blood-derived cells can differentiate into hepatocytes in the mouse liver with no evidence of cellular fusion. Gastroenterology 124: 1891–1900, 2003.[CrossRef][Web of Science][Medline]
  28. Okuyma T, Fujino M, Li XK, Funeshima N, Kosuga M, Saito I, Suzuki S, and Yamada M. Efficient Fas-ligand gene expression in rodent liver after intravenous injection of a recombinant adenovirus by the use of a Cre-mediated switching system. Gene Ther 5: 1047–1053, 1998.[CrossRef][Web of Science][Medline]
  29. Peled A, Petit I, Kollet O, Magid M, Ponomaryov T, Byk T, Nagler A, Ben-Hur H, Many A, Shultz L, Lider O, Alon R, Zipori D, and Lapidot T. Dependence of human cell engraftment and repopulation of NOD/SCID mice on CXCR4. Science 283: 845–848, 1999.[Abstract/Free Full Text]
  30. Petersen BE, Bowen WC, Patrene KD, Mars WM, Sullivan AK, Murasa N, Boggs SS, Greenberger JS, and Goff JP. Bone marrow as a potential source of hepatic oval cells. Science 284: 1168–1170, 1999.[Abstract/Free Full Text]
  31. Petersen BE, Goff JP, Greenberger JS, and Michalopoulos G. Hepatic oval cells express the hematopoietic stem cell marker Thy-1 in the rat. Hepatology 27: 433–445, 1998.[CrossRef][Web of Science][Medline]
  32. Roche V, Wagner JE, Sobocinski KA, Klein JP, Zhang MJ, Horowitz MM, and Gluckman E. Graft-versus-host disease in children who have received a cord-blood or bone marrow transplant from an HLA-identical sibling. Eurocord and International bone marrow transplant registry working committee on alternative donor and stem cell sources. N Engl J Med 342: 1846–1854, 2000.[Abstract/Free Full Text]
  33. Theise ND, Nimmakayalu M, Gardner R, Illei PB, Morgan G, Teperman L, Henegariu O, and Krause DS. Liver from bone marrow in humans. Hepatology 32: 11–16, 2000.[CrossRef][Web of Science][Medline]
  34. Theise ND, Saxena R, Portmann BC, Thung SN, Yee H, Chiriboga L, Kumar A, and Crawford JM. The canal of Hering and hepatic stem cells in humans. Hepatology 30: 1425–1433, 1999.[CrossRef][Web of Science][Medline]
  35. Ueda T, Yoshino H, Kobayashi K, Kawahata M, Ebihara Y, Ito M, Asano S, Nakahata T, and Tsuji K. Hematopoietic repopulationing ability of cord blood CD34+ cells in NOD/SCID mice. Stem Cells 18: 204–213, 2000.[Abstract/Free Full Text]
  36. Vassilopoulos G, Wang PR, and Russell DW. Transplanted bone marrow regenerates liver by cell fusion. Nature 422: 901–904, 2003.[CrossRef][Medline]
  37. Vessey CJ and de la Hall PM. Hepatic stem cells: a review. Pathology 33: 130–141, 2001.[Web of Science][Medline]
  38. Wagers AJ, Sherwood RI, Christensen JL, and Weissman IL. Little evidence for development plasticity of adult hematopoietic stem cells. Science 297: 2256–2259, 2002.[Abstract/Free Full Text]
  39. Wang JCY, Doedens M, and Dick JE. Primitive human hematopoietic cells are enriched in cord blood compared with adult bone marrow or mobilized peripheral blood as measured by the quantitative in vivo SCID-repopulating cell assay. Blood 89: 3919–3924, 1997.[Abstract/Free Full Text]
  40. Wang X, Ge S, McNamara G, Hao QL, Crooks GM, and Nolta JA. Alubumin-expressing hepatocyte-like cells develop in the livers of immune-deficient mice that received transplants of highly purified human hematopoietic stem cells. Blood 101: 4201–4208, 2003.[Abstract/Free Full Text]
  41. Wang X, Willenbring H, Akkari Y, Torimaru Y, Foster M, Al-Dhalimy M, Lagasse E, Finegold M, Olson S, and Grompe M. Cell fusion is the principal source of bone-marrow-derived hepatocytes. Nature 422: 897–901, 2003.[CrossRef][Medline]
  42. Willenbring H, Bailey AS, Foster M, Akkari Y, Dorrell C, Olson S, Finegold M, FlemingWH, and Grompe M. Myelomonocytic cells are sufficient for therapeutic cell fusion in liver. Nat Med 10: 744–748, 2004.[CrossRef][Web of Science][Medline]
  43. Yamamoto N, Terai S, Ohata S, Watanabe T, Omori K, Shinoda K, Miyamoto K, Katada T, Sakaida I, Nishina H, and Okita K. A subpopulation of bone marrow cells depleted by a novel antibody, anti-Liv8, is useful for cell therapy to repair damaged liver. Biochem Biophys Res Commun 313: 1110–1118, 2004.[CrossRef][Web of Science][Medline]
  44. Zecca M, Prete A, Rondelli R, Lanino E, Balduzzi A, Messina C, Fagioli F, Porta F, Favre C, Pession A, and Locatelli F. Chronic graft-versus-host disease in children: incidence, risk factors, and impact on outcome. Blood 100: 1192–1200, 2002.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 GutHome page
P Sancho-Bru, M Najimi, M Caruso, K Pauwelyn, T Cantz, S Forbes, T Roskams, M Ott, U Gehling, E Sokal, et al.
Stem and progenitor cells for liver repopulation: can we standardise the process from bench to bedside?
Gut, April 1, 2009; 58(4): 594 - 603.
[Abstract] [Full Text] [PDF]

Home page
 DMMHome page
N. Brezillon, D. Kremsdorf, and M. C. Weiss
Cell therapy for the diseased liver: from stem cell biology to novel models for hepatotropic human pathogens
Dis. Model. Mech., September 1, 2008; 1(2-3): 113 - 130.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
289/6/G1091    most recent
00049.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (8)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Nonome, K.
Right arrow Articles by Sugiyama, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Nonome, K.
Right arrow Articles by Sugiyama, T.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2005 by the American Physiological Society.