Embryonic hepatic stem cells from GFP transgenic mice: in vitro separation, cultivation and intra-spleen transplantation

doi:10.3969/j.issn.2095-4344.0910

Abstract

Abstract:

BACKGROUND: Recent studies have confirmed that the success of stem cell transplantation and the function of transplanted cells are closely related to the in vivo microenvironment. Under normal physiological conditions, the liver is not susceptible to exogenous cells. Therefore, a transplant animal model is usually established to damage the host liver or reduce the immune response of the host liver, creating a good condition for the transplantation and proliferation of exogenous cells.
OBJECTIVE: To observe the colonization and distribution of embryonic hepatic stem cells from GFP transgenic mice in a rat model of liposome-coated dichloromethylene diphosphate/retrorsine and 1/3 liver resection (L-CL₂MDP/RS/PH).
METHODS: GFP transgenic mice at gestational 15 days were taken as donors to separate and cultivate embryonic hepatic stem cells. Fluorescence microscope was used to observe the GFP expression followed by immunocytochemical identification. Thirty Sprague-Dawley rats were randomized into experimental group and control group. Rats in the experimental group were given intraperitoneal injection of retrorsine (30 mg/kg, twice, with an interval of 2 weeks). At 1 preoperative day, these rats were subjected to intravenous infusion of L-CL₂MDP (1 mL) and 1/3 liver resection followed by intra-spleen transplantation of embryonic hepatic stem cells from GFP transgenic mice. Rats in the control group were only given injection of normal saline instead of L-CL₂MDP and retrorsine, but no liver resection. Frozen liver sections were made in the two groups to observe the distribution of GFP-positive hepatic stem cells under the fluorescence microscope at 3, 15, 30 days after transplantation. Moreover, immunohistochemical staining was used to detect the expression of anti-mouse C-Kit antibody in the rat liver.
RESULTS AND CONCLUSION: (1) The hepatic stem cells under microscope were uniform with a cobblestone shape. After 5 days of culture, the cells with increased size presented with the shape of fusiform or polygon, and the green fluorescence was observed in cells under the fluorescence microscope. Passage 3 hepatic stem cells expressed CD34 and AFP. (2) At 3 days after spleen transplantation of embryonic hepatic stem cells, GFP positive cells were scattered in the experimental group under the fluorescence microscope, and the GFP positive expression was still visible in the liver at 30 days after transplantation. (3) At 3, 15, and 30 days after transplantation, C-Kit positive cells were visible in the liver of rats in the experimental group. All these findings reveal that embryonic hepatic stem cells from GFP transgenic mice have been successfully transplanted into the spleen of the L-CL₂MDP/RS/PH rat model, and the transplanted cells can survive and proliferate in the rat liver.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Liver, Stem Cells, Mice, Transgenic, Tissue Engineering

CLC Number:

R394.2

Wang Ya-ping, Kong Xiang-ping, Liu Guang-ze, Zhuo Li, Gao Hong-bo, Tang Xiao-ping, Tong Ming-hua. Embryonic hepatic stem cells from GFP transgenic mice: in vitro separation, cultivation and intra-spleen transplantation[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(25): 4071-4076.

Figures/Tables 2

References

[1] Diehl AM, Rai R. Review: regulation of liver regeneration by pro-inflammatory cytokines. J Gastroenterol Hepatol. 1996; 11(5):466-470.

[2] Banas A, Teratani T, Yamamoto Y, et al. Rapid hepatic fate specification of adipose-derived stem cells and their therapeutic potential for liver failure. J Gastroenterol Hepatol. 2009;24(1):70-77.

[3] Yu Y, Yao AH, Chen N, et al. Mesenchymal stem cells over-expressing hepatocyte growth factor improve small-for-size liver grafts regeneration. Mol Ther. 2007;15(7): 1382-1389.

[4] Pournasr B, Mohamadnejad M, Bagheri M, et al. In vitro differentiation of human bone marrow mesenchymal stem cells into hepatocyte-like cells. Arch Iran Med. 2011;14(4): 244-249.

[5] Oyagi S, Hirose M, Kojima M, et al. Therapeutic effect of transplanting HGF-treated bone marrow mesenchymal cells into CCl4-injured rats. J Hepatol. 2006;44(4):742-748.

[6] Si-Tayeb K, Noto FK, Nagaoka M, et al. Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem cells. Hepatology. 2010;51(1):297-305.

[7] Yu LM, Luo N, Li QL, et al. EPCAM-positive normal hepatic progenitor cells transformation into liver stem cells and HBx-mediated effects on stability in adult mouse. Zhonghua Gan Zang Bing Za Zhi. 2015;23(11):854-859.

[8] Giri S, Acikgöz A, Bader A. Isolation and Expansion of Hepatic Stem-like Cells from a Healthy Rat Liver and their Efficient Hepatic Differentiation of under Well-defined Vivo Hepatic like Microenvironment in a Multiwell Bioreactor. J Clin Exp Hepatol. 2015;5(2):107-122.

[9] Habeeb MA, Vishwakarma SK, Bardia A, et al. Hepatic stem cells: A viable approach for the treatment of liver cirrhosis. World J Stem Cells. 2015;7(5):859-865.

[10] Khan AA, Shaik MV, Parveen N, et al. Human fetal liver-derived stem cell transplantation as supportive modality in the management of end-stage decompensated liver cirrhosis. Cell Transplant. 2010; 19(4):409-418.

[11] 吴波,张博,李伟光,等.TGF-β1基因修饰的肝干细胞诱导大鼠移植肝脏免疫耐受的实验研究[J].胃肠病学和肝病学杂志, 2014, 23(4):425-427.

[12] 韩博,徐三荣,张进,等.同基因与异基因胚胎肝干细胞移植治疗小鼠肝硬化[J].中国组织工程研究,2013,16(36):6474-6480.

[13] 张锡峰,胡代曦,芦永良,等. Wnt3a过表达对小鼠胚胎肝干细胞凋亡的抑制作用[J].细胞与分子免疫学杂志, 2013,29(12): 1277-1280.

[14] 黄增辉,曾珊,欧阳淼,等.肝素联合肝干细胞经脾移植治疗SD大鼠急性肝损伤[J].中南大学学报:医学版,2011,36(5):411-416.

[15] Ito M, Nagata H, Miyakawa S, et al. Review of hepatocyte transplantation. J Hepatobiliary Pancreat Surg. 2009;16(2): 97-100.

[16] Carpentier B, Gautier A, Legallais C. Artificial and bioartificial liver devices: present and future. Gut. 2009;58(12): 1690-1702.

[17] 刘勇,冯东福,陈二涛,等.绿色荧光蛋白转基因胚胎大鼠神经干细胞生物学特性研究[J].实用医学杂志,2008,24(5):709-712.

[18] Gai H, Nguyen DM, Moon YJ, et al. Generation of murine hepatic lineage cells from induced pluripotent stem cells. Differentiation. 2010;79(3):171-181.

[19] Gouon-Evans V, Boussemart L, Gadue P, et al. BMP-4 is required for hepatic specification of mouse embryonic stem cell-derived definitive endoderm. Nat Biotechnol. 2006;24(11): 1402-1411.

[20] Polisetti N, Chaitanya VG, Babu PP, et al. Isolation, characterization and differentiation potential of rat bone marrow stromal cells. Neurol India. 2010;58(2):201-208.

[21] Sakaida I. Autologous bone marrow cell infusion therapy for liver cirrhosis. J Gastroenterol Hepatol. 2008;23(9): 1349-1353.

[22] 周晓飞,王茜,褚建新,等. Retrorsine对小鼠肝损伤后肝细胞增殖的影响[J].第二军医大学学报,2006,27(1):31-35.

[23] Mohamadnejad M, Alimoghaddam K, Mohyeddin-Bonab M, et al. Phase 1 trial of autologous bone marrow mesenchymal stem cell transplantation in patients with decompensated liver cirrhosis. Arch Iran Med. 2007;10(4):459-466.

[24] Giuliani AL, Wiener E, Lee MJ, et al. Changes in murine bone marrow macrophages and erythroid burst-forming cells following the intravenous injection of liposome-encapsulated dichloromethylene diphosphonate (Cl2MDP). Eur J Haematol. 2001;66(4):221-229.

[25] Bauer D, Schmitz A, Van Rooijen N, et al. Conjunctival macrophage-mediated influence of the local and systemic immune response after corneal herpes simplex virus-1 infection. Immunology. 2002;107(1):118-128.

[26] Tsai PC, Fu TW, Chen YM, et al. The therapeutic potential of human umbilical mesenchymal stem cells from Wharton's jelly in the treatment of rat liver fibrosis. Liver Transpl. 2009; 15(5):484-495.

[27] Levicar N, Pai M, Habib NA, et al. Long-term clinical results of autologous infusion of mobilized adult bone marrow derived CD34+ cells in patients with chronic liver disease. Cell Prolif. 2008;41 Suppl 1:115-125.

[28] 李劲,张健,刘光泽,等. D-氨基半乳糖与脂多糖对小鼠肝脏损伤后再生修复的影响[J].南方医科大学学报,2012,32(1):50-54.