Abstract




BACKGROUND: In vitro differentiation of embryonic stem cells into hepatocytes has been successfully reported to a certain degree; however, whether embryonic stem cells are able to effectively enter hepatic plate of host after intrahepatic transplantation, whether embryonic stem cells can further differentiate into hepatocytes and express hepatocyte function, and risk factors for neoplastic formation are still unclear at present.
OBJECTIVE: To study the intrahepatic transplantation of embryonic stem cells-derived hepatic stem cells in therapeutic liver repopulation models, and to investigate the liver tissue replacement, growth and differentiation in vivo, and neoplastic formation.
DESIGN: Randomized controlled animal study.
SETTING: Department of Pediatric Surgery, the Second Hospital affiliated to Sun Yat-sen University.
MATERIALS: Twenty-four BALB/c mice, 6-8 weeks old, weighing 20-35 g, irrespective of gender, were provided by Guangzhou Experimental Animal Center. Embryonic stem cells-derived hepatic stem cells were differentiated from embryonic stem cells. E14 was provided by Stem cell Center of our hospital.
METHODS: This study was performed at the Stem Cell Center, the Second Hospital affiliated to Sun Yat-sen University from July 2006 to June 2007. Twenty-four mice were randomly divided into a liver repopulation model + stem cell transplantation group (group A) and a liver resection + stem cell transplantation group (group B), with 12 mice in each group. Mice in the group A were intraperitoneally injected with 50 mg/kg retrorsine once every two weeks for totally twice. Four weeks after the second injection, about 70% liver was resected. And then, the embryonic stem cells-derived hepatic stem cells, labeled by 1×105 carboxy fluoresce in diacetate succinimidyl ester (CFDA-SE), were transplanted into mouse liver through portal vein. On the other hand, 70% liver of mice in the group B was resected and embryonic stem cells-derived hepatic stem cells were transplanted into mouse liver.
MAIN OUTCOME MEASURES: The distribution, incorporation, and proliferation of transplanted cells were observed under fluorescent microscopy. Two weeks later, hepatic function was stained with albumin fluorescence immunoassay (double fluorescence staining) and assayed by level of serum albumin. Embryonic stem cells-derived hepatic stem cells were poured into liver of remedial liver regeneration mice, and undifferentiated embryonic stem cells were transplanted into subcutaneous tissue in axillary region as the controls to observe neoplastic formation in embryonic stem cells-derived hepatic stem cells.
RESULTS: ① Growth of hepatic stem cells in recipient mice: One week after transplantation of CFDA-SE-labeled embryonic stem cells-derived hepatic stem cells, some scattered region was green under fluorescent microscopy. The area of green region increased apparently in 2 weeks, and cord-like structure could be observed. ② Liver function: Immunofluorescent staining of albumin (double fluorescence staining) demonstrated that labeled cells expressed positive albumin (yellow fluorescence) in liver tissue of recipient mice, but there was not significant difference in serum albumin level between group A and group B (P > 0.05). ③ Reliability of hepatic stem cell transplantation: Teratoma did not form over 6 months; however, transplantation of undifferentiated embryonic stem cells in the axillary region could cause formation of teratoma after 6 weeks.
CONCLUSION: The transplantation of embryonic stem cells-derived hepatic stem cells in therapeutic liver repopulation model mice can effectively and further grow and differentiate, or even partially express hepatocyte function; in particular, the transplantation is safe.

INTRODUCTION

In vitro differentiation of embryonic stem cells into hepatocytes has been successfully reported to a certain degree; however, whether embryonic stem cells are able to effectively enter hepatic plate of host after intrahepatic transplantation, whether embryonic stem cells can further differentiate into hepatocytes and express hepatocyte function, and risk factors for neoplastic formation are still unclear at present[1-8]. Previous researches proved that in vitro culture adding fibroblast growth factor-4 (FGF-4), hepatocyte growth factor (HGF) and dexamethasone (Dex) during liver embryogeny can induce embryonic stem cells to differentiate into hepatocyte-like cells. A pathological microenvironmental culture system which consists of cholestatic serum can not only effectively screen hepatocyte-like cells with partial liver function from embryonic stem cells initially induced by FGF-4 and HGF, but also further promote the growth and differentiation of hepatocyte-like cells[9-11].
In this study, hepatocyte-like cells[1] screened using the pathological microenvironmental culture system consisting of cholestatic serum were transplanted into liver of therapeutic liver repopulation model mice through portal vein to determine whether hepatocyte-like cells could effectively integrate into hepatic plate of host and whether hepatocyte-like cells could further grow, differentiate, and express hepatocyte function. On the other hand, neoplastic formation was analyzed to evaluate reliability of the screened embryonic stem cells-derived hepatic stem cells.

MATERIALS AND METHODS

Materials
This study was performed at the Stem Cell Center, the Second Hospital affiliated to Sun Yat-sen University from July 2006 to June 2007. Twenty-four BALB/c mice, 6-8 weeks old, weighing 20-35 g, irrespective of gender, clean grade, were provided by Guangzhou Experimental Animal Center. E14 was provided by Stem cell Center of our hospital. Embryonic stem cells-derived hepatic stem cells were differentiated from embryonic stem cells. A pathological microenvironmental culture system consisting of cholestatic serum was used to effectively screen hepatocyte-like cells with partial liver function[1,9].

Methods
Model establishment and animal grouping
Twenty-four mice were randomly divided into a liver repopulation model + stem cell transplantation group (group A) and a liver resection + stem cell transplantation group (group B), with 12 mice in each group. Mice in the group A were intraperitoneally injected with 50 mg/kg retrorsine once every two weeks for totally twice. Four weeks after the second injection, about 70% liver was resected. And then, the embryonic stem cells-derived hepatic stem cells, labeled by 1×105 carboxy fluoresce in diacetate succinimidyl ester (CFDA-SE), were transplanted into mouse liver through portal vein. On the other hand, 70% liver of mice in the group B was resected and embryonic stem cells-derived hepatic stem cells were transplanted into mouse liver.

Intrahepatic transplantation of embryonic stem cells-derived hepatic stem cells
CFDA-SE (Molecular Probes) was used to label the transplanted cells: ① Two-week-cultured cells were digested and screened with 0.25% pancreatin and centrifuged at 1 000 r/min for 5 minutes. And then, the supernatant was removed, and the cells were re-suspended in PBS containing 10 μmol/L CFDA-SE and incubated at 37 ℃ for 15 minutes to make sure the complete absorption of fluorescent tracer. ② The obtained cells were centrifuged at 1 000 r/min for 5 minutes. And then, the supernatant was removed, and the cells were re-suspended in PBS and incubated at 37 ℃ for 30 minutes. Acetyl group was removed from CFDA-SE under the action of esterase in plasma, and CFDA-SE was fluorescent. ③ The samples were observed and labeled under fluorescent microscopy, and labeling rate was detected using flow cytometer. Cells were diluted with PBS to the concentration of 1×109 cells/L. Mice were anesthetized with diethyl ether to expose the abdomens. Blood flow of portal vein was blocked for 15 minutes by using forceps clip, and then fluorescence-labeled cells (1×105) were transplanted into mouse liver through portal vein.

Differentiation in vivo
One, three days, one and two weeks after transplantation, liver tissue samples were observed under fluorescent microscopy (×100 or ×200) to detect in vivo distribution, integration, replacement, growth, and differentiation of transplanted cells.

Detection of liver function
Differentiation of donor stem cells was determined in recipient liver by using albumin fluorescence immunoassay (double fluorescence staining). ① Basic principle: Albumin is a functional protein of hepatocyte-specific expression, and also a functional indicator of mature hepatocyte. Its expression quantity could directly reflect differentiated degree and functional status of transplanted stem cells. It was hard to identify hepatocytes differentiated from donor stem cells based on form and function in recipient liver tissue. Transplantation of green fluorescent protein CFDA-SE-labeled donor cells helped show distribution of donor cells in recipient liver tissue; while red fluorescent protein-labeled albumin immunoassay helped evaluate differentiation of donor stem cells. ② Experimental procedures: Two weeks after transplantation of embryonic stem cells-derived hepatic stem cells, three mice in each group were anesthetized and sacrificed. Liver tissue was rapidly obtained, frozen, and cut into serial sections (5 μm in thickness). The frozen sections were fixed with 4% formaldehyde, defrosted at room temperature for 15- 30 minutes, and hydrated with PBS at room temperature in wet box for 15 minutes. And then, the samples were added with PBS containing 0.2% Triton X-100 (PBT) and maintained at room temperature for 20 minutes, added with PBT containing 0.2% BSA and 5% goat serum (or 1% BSA) and maintained at room temperature for 30 minutes (to block non-specific binding), added with 1 μg antibody Ⅰ (rabbit-anti-mouse albumin monoclonal antibody dissolved in 20 μL PBS) and 5 μL antibody Ⅱ (Zenon A reagent dissolved in antibody Ⅰ solution) and incubated at room temperature for 5 minutes (albumin antibody was labeled by PE), and added with 5 μL Zenon B reagent and incubated at room temperature for 5 minutes to block labeling reaction. PBT (3:1) was used to dissolve fluorescent-labeled albumin antibody (immune compound of antibodies Ⅰ and Ⅱ). Tissue sections were dripped with fluorescent-labeled albumin antibody and incubated in dark slide at room temperature for 1.0-2.0 hours. Subsequently, the samples were washed with PBT twice with each for 10-15 minutes and with PBS twice with each for 5 minutes, re-fixed with 4% formaldehyde at room temperature for 15 minutes, and washed with PBS. Finally, the samples were put under confocal fluorescent microscope to observe fluorescent signals.
Two weeks after transplantation, blood was taken by using routine biochemical check method to measure serum albumin level.

Reliability evaluation
Embryonic stem cells-derived hepatic stem cells were poured into liver of therapeutic liver repopulation mice, and undifferentiated embryonic stem cells were transplanted into subcutaneous tissue in axillary region as the controls to observe neoplastic formation in embryonic stem cells-derived hepatic stem cells over 6 weeks.

Statistical analysis
Statistical analysis was manually calculated by the first author. Measurement data were compared with t test.

RESULTS

Intrahepatic distribution of CFDA-SE-labeled cells after transplantation in recipient mice
In one week after transplantation of CFDA-SE-labeled embryonic stem cells-derived hepatic stem cells in recipient mice, scattered green fluorescence was distributed in hepatic parenchyma in both groups, and density was generally coincident (Figure 1a). Two weeks later, scattered green fluorescence was enlarged in hepatic parenchyma in group A, and the scattered green fluorescence arrayed like hepatic cord structure (Figure 1b). The area of scattered green fluorescence in the group A was not significantly enlarged as compared in the group B, but a hepatic cord-like structure was still observed (Figure 1c).
Normal liver tissue was red under fluorescent microscopy, while CFDA-SE-labeled cells were green under fluorescent microscopy. The labeled cells expressed albumin were red and green (double fluorescence staining), i.e., yellow under fluorescent microscopy. Two weeks after transplantation of CFDA-SE-labeled embryonic stem cells-derived hepatic stem cells, albumin fluorescent immunohistochemistry assay demonstrated that labeled cells could express positive signals of albumin in liver tissue of recipient mice in the two groups (Figure 1d). However, amount of positive signals in the group A was apparently higher than in the group B.

Liver function
Two weeks after transplantation of hepatic stem cells, serum albumin levels were (29.9±1.45) g/L in the group A and (29.2±1.93) g/L in the group B, and there was not significant difference (P > 0.05).

Reliability evaluation
Transplantation of undifferentiated embryonic stem cells in the axillary region could cause formation of teratoma after 6 weeks (Figure 2); however, pouring hepatocyte-like cells screened by pathological microenvironmental culture system into remedial liver regeneration model mice could not cause the formation of teratoma in 6 weeks.

DISCUSSION

Therapeutic liver repopulation technique helps injured hepatocytes completely replace by transplanted cells through only a few of hepatocyte transplantation to achieve the same replacement effect of a whole organ through orthotopic liver transplantation (OLT). At normal condition, hepatocytes of recipient itself proliferates precedently, so transplanted hepatocytes are not able to proliferate to reach the required amount. Otherwise, if retrorsine (RS) is used to pre-inhibit proliferation of recipient hepatocytes, followed by applying exogenous factors which can promote growth and differentiation of hepatocytes or stimulating excretion of exogenous factors through inducing liver injury, transplanted hepatocytes will be promoted to proliferate and nearly completely replace original hepatocytes[12-17]. Therefore, this study was designed to establish remedial liver regeneration models for transplanting hepatocyte-like cells screened by using pathological microenvironmental culture system through portal vein to determine whether hepatocyte-like cells could effectively integrate into hepatic plate of host and whether hepatocyte-like cells could further grow, differentiate, and express hepatocyte function. On the other hand, neoplastic formation was analyzed to evaluate reliability of the screened embryonic stem cells-derived hepatic stem cells.
How to differentiate transplanted cells among a lot of hepatocytes is still to be solved recently. Positive cells of Y chromatosome is detected with fluorescence in situ hybridization (FISH) by using cross sex transplantation technique[6], or gene knock-out mouse model is used by some scholars[18-19]; however, these methods are highly restricted by experimental conditions. Therefore, it is necessary to establish a new labeling method. We found that CFDA-SE-labeled cell differentiation was passable. CFDA-SE is a compound of carboxy fluoresce in diacetate and succinimidyl ester, and it does not have fluorescence outside soma. Being in cells, CFDA-SE takes off acetyl and shows green fluorescence under the action of esterase; in addition, it combines with amino group into fluorescence compound, which can label cells for a long period and transfer with the cell division. So it is a good cell tracer[20]. Hepatocyte specific protein, for example, albumin, will be used for background staining (double fluorescence staining) to indicate distribution of transplanted cells or changes of liver structure.
This study suggested that in one week after transplantation of CFDA-SE-labeled embryonic stem cells-derived hepatic stem cells in recipient mice, scattered green fluorescence was distributed in hepatic parenchyma in both groups, and density was generally coincident. Two weeks later, scattered green fluorescence was enlarged in hepatic parenchyma in group A, and the scattered green fluorescence arrayed like hepatic cord structure. The area of scattered green fluorescence in the group A was not significantly enlarged as compared in the group B, but a hepatic cord-like structure was still observed. Normal liver tissue was red under fluorescent microscopy, while CFDA-SE-labeled cells were green under fluorescent microscopy. The labeled cells expressed albumin were red and green (double fluorescence staining), i.e., yellow under fluorescent microscopy. Two weeks after transplantation of CFDA-SE-labeled embryonic stem cells-derived hepatic stem cells, albumin fluorescent immunohistochemistry assay demonstrated that labeled cells could express positive signals of albumin in liver tissue of recipient mice in the two groups. However, amount of positive signals in the group A was apparently higher than in the group B. Transplantation of undifferentiated embryonic stem cells in the axillary region could cause formation of teratoma after 6 weeks; however, pouring hepatocyte-like cells screened by pathological microenvironmental culture system into therapeutic liver repopulation model mice could not cause the formation of teratoma in 6 weeks.
In a word, the transplantation of embryonic stem cells-derived hepatic stem cells in therapeutic liver repopulation model mice can effectively integrate and enter hepatic plate of host, and embryonic stem cells-derived hepatic stem cells can further grow and differentiate or even express hepatocyte function; in particular, the transplantation is safe, and teratoma is not formed in 6 weeks. This study rudimentally tries to internally transplant embryonic stem cells-derived hepatic stem cells in remedial liver regeneration models, but how to rebuilt a liver organ with full function using embryonic stem cells still needs a further study.

REFERENCES

1 Fang S, Qiu YD, Mao L, et al. Differentiation of embryoid-body cells derived from embryonic stem cells into hepatocytes in alginate microbeads in vitro.Acta Pharmacol Sin 2007;28(12):1924-1930
2 Alison MR, Choong C, Lim S.Application of liver stem cells for cell therapy.Semin Cell Dev Biol 2007;18(6):819-826
3 Cho CH, Parashurama N, Park EY, et al. Homogeneous differentiation of hepatocyte-like cells from embryonic stem cells: applications for the treatment of liver failure.FASEB J （in press）
4 Banas A, Yamamoto Y, Teratani T, et al. Stem cell plasticity: Learning from hepatogenic differentiation strategies.Dev Dyn 2007;236(12):3228-3241
5 Ishii T, Yasuchika K, Machimoto T, et al. Transplantation of embryonic stem cell-derived endodermal cells into mice with induced lethal liver damage.Stem Cells 2007;25(12):3252-3260
6 Duan Y, Catana A, Meng Y, et al. Differentiation and enrichment of hepatocyte-like cells from human embryonic stem cells in vitro and in vivo.Stem Cells 2007;25(12):3058-3068
7 Zhou QJ, Huang YD, Xiang LX, et al. In vitro differentiation of embryonic stem cells into hepatocytes induced by fibroblast growth factors and bone morphological protein-4.Int J Biochem Cell Biol 2007;39(9):1714-1721
8 Cai J, Zhao Y, Liu Y, et al. Directed differentiation of human embryonic stem cells into functional hepatic cells.Hepatology 2007;45(5):1229-1239
9 Deng XG, Fang TL, Cao MH, et al. Differentiation mouse ES cells into hepatocytes induced by a pathological microenvironmental culture system consisting of cholestatic sera. Zhongguo Bingli Shengli Zazhi 2004;20(6):985-989
10 Deng XG, Min J, Zhang J, et al. Induced differentiation mouse ES cells into hepatocytes in vitro.　Zhonghua Xiaoer Waike Zazhi 2006;27（6）：313-317
11 Min J, Shang CZ, Chen YJ, et al. Selective enrichment of hepatocytes from mouse embryonic stem cells with a culture system containing cholestatic serum. Acta Pharmacol Sin 2007;28(12):1931-1937
12 Grompe M. Therapeutic liver repopulation for the treatment of metabolic liver diseases. Hum Cell 1999;12(4):171-180
13 Grompe M, Laconi E, Shafritz DA. Principles of therapeutic liver repopulation. Semin Liver Dis 1999;19(1):7-14
14 Laconi S, Pillai S, Porcu PP, et al. Massive liver replacement by transplanted hepatocytes in the absence of exogenous growth stimuli in rats treated with retrorsine.Am J Pathol 2001;158(2):771-777
15 Grompe M. Principles of therapeutic liver repopulation. J Inherit Metab Dis 2006;29(2-3):421-425
16 Wang X, Foster M, Al-Dhalimy M, et al. The origin and liver repopulating capacity of murine oval cells.Proc Natl Acad Sci U S A 2003;100 Suppl 1:11881-11888
17 Weissman IL. Translanting stem and progeniter cell biology to the clinic: barriers and opportunities. Science 2000;287( 5457): 1442-1446
18 Dabeva MD, Petkov PM, Sandhu J, et al. Proliferation and differentiation of fetal liver epithelial progenitor cells after transplantation into adult rat liver. Am J Pathol 2000;156(6):2017-2031
19 Sandhu JS, Petkov PM, Dabeva MD, et al. Stem cell properties and repopulation of the rat liver by fetal liver epithelial progenitor cells. Am J Pathol 2001,159(4):1323-1334
20 Mueller-Stahl K, Kofidis T, Akhyari P, et al. Carboxyfluorescein diacetate succinimidyl ester facilitates cell tracing and colocallization studies in bioartificial organ engineering. Int J Artif Organs 2003;26(3): 235-240

胚胎干细胞源性肝干细胞肝内移植对
宿主肝功能的影响及安全性评估**☆

邓小耿， 宋尔卫，闵 军，张 杰，陈 伦，曾炳胜，方天翎， 陈积圣
中山大学附属第二医院小儿外科， 广东省广州市 510120
邓小耿☆，男，1968年生，湖南省汝城县人，汉族，2004年中山大学毕业，博士，副主任医师，主要从事干细胞诱导分化的研究。
国家自然科学基金（30471799）*；广东省自然科学基金资助项目（06300736）*
摘要

背景：体外诱导胚胎干细胞分化为肝细胞已有不少成功的报道，但其体内移植后能否有效整合入宿主肝板、在肝内能否进一步生长分化并表达肝细胞功能以及成瘤的风险等情况目前还不清楚。
目的：应用治疗性肝再生模型进行胚胎干细胞源性肝干细胞肝内移植, 观察其在肝组织替代、体内的生长分化及成瘤性情况。

设计：随机对照动物实验。
单位：中山大学附属第二医院小儿外科。
材料：选用BALB/c小鼠24只为受体，鼠龄6~8周，体质量20~ 35 g，雌雄不拘购自广州市实验动物中心。实验所用胚胎干细胞源性肝干细胞由作者所在课题组诱导胚胎干细胞分化而成。小鼠胚胎干细胞株E14由本院干细胞中心提供。
方法：实验于2006-07/2007-06在中山大学附属第二医院干细胞研究中心完成。将24只小鼠随机分为2组：肝再生模型+干细胞移植组和肝切除+干细胞移植组，每组12只。前组分两次按50 mg/kg剂量腹腔内注射倒千里光碱(retrorsine) ，间隔2周，第2次注射4周后行70%肝部分切除制造肝损伤；然后经门静脉分别移植1×105羟基荧光素乙酰乙酸（CFDA-SE）荧光标记的细胞入小鼠肝内进行胚胎干细胞源性肝干细胞移植。后组在行70%肝部分切除制造肝损伤模型后进行胚胎干细胞源性肝干细胞移植。
主要观察指标：荧光显微镜下观察移植细胞组受体鼠肝脏内分布、整合与体内生长分化情况。2周后行白蛋白荧光免疫组化(双荧光染色)、血清白蛋白水平检测其功能状况。将胚胎干细胞源性肝干细胞注入治疗性肝再生小鼠肝内，将未分化的胚胎干细胞移植入小鼠腋区皮下作为对照，观察胚胎干细胞源性肝干细胞体内成瘤情况。
结果：①肝干细胞在受体鼠肝内生长情况：CFDA SE标记的胚胎干细胞源性肝干细胞肝内移植1周，受体小鼠肝实质内可见散在绿色荧光分布。2周后，肝实质内绿色荧光分布区域明显扩大，且可见类似肝索样结构排列。②肝功能：共焦白蛋白荧光免疫组化(双荧光染色)结果表明，受体小鼠肝组织内可见标记细胞表达白蛋白阳性信号（呈黄色荧光），肝再生模型+干细胞移植组和肝切除+干细胞移植组血清白蛋白水平则无明显差异（P > 0.05）。③肝干细胞移植安全性：6周内未见畸胎瘤形成，而将未分化的胚胎干细胞移植入小鼠腋区皮下6周后则可见畸胎瘤形成。
结论：胚胎干细胞源性肝干细胞移植入治疗性肝再生模型小鼠肝内后可有效在肝内能进一步生长分化并部分表达肝细胞功能；且此移植安全性较好。
关键词：胚胎干细胞；肝细胞移植；诱导分化；治疗性肝再生；淤胆血清

中图分类号: R394.2 文献标识码: A 文章编号: 1673-8225(2008)08-01591-05
邓小耿， 宋尔卫，闵军，张杰，陈伦，曾炳胜，方天翎，陈积圣.胚胎干细胞源性肝干细胞肝内移植对宿主肝功能的影响及安全性评估[J].中国组织工程研究与临床康复，2008，12(8):1591-1595
[www.zglckf.com/zglckf/ejournal/upfiles/08-8/8k-1591(ps).pdf]
(Edited by Jiang JY/Ji H/Wang L)