Proliferation of Hepatocyte Progenitor Cells Isolated from Adult Human Livers in Serum-Free Medium
Proliferation of Hepatocyte Progenitor Cells Isolated from Adult Human Livers in Serum-Free Medium
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DOI：
10.3727/096368908787236666
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发表时间：
2008-10
影响因子：
3.3
通讯作者：
K. Sasaki;J. Kon;T. Mizuguchi;Qijie Chen;Hidekazu Ooe;H. Oshima;K. Hirata;T. Mitaka
中科院分区：
文献类型：
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作者：
K. Sasaki;J. Kon;T. Mizuguchi;Qijie Chen;Hidekazu Ooe;H. Oshima;K. Hirata;T. Mitaka
大鼠小肝细胞(SHS)是一种定向祖细胞，在透明质酸(HA)包被的培养皿中可以分化为成熟的肝细胞，并在无血清介质中选择性增殖。在这项研究中，我们研究了人类SHS从成人肝脏中分离的情况。我们从16例接受肝切除的患者的切除肝中获取肝组织。提取的肝脏标本与肿瘤区域明显分离，边缘充足。采用改良的二步胶原酶灌注法分离肝细胞。采用低速离心法，将培养上清中的细胞接种于含烟酰胺、表皮生长因子和肝细胞生长因子的DMEM/F12无血清培养皿中。小肝细胞选择性增殖形成集落，许多集落持续生长3周以上。培养第7天、第14天和第21天的集落平均细胞数分别为38.6±18.0、79.0±54.0和101.5±115.7个。约0.04%的平板细胞可形成SH集落。免疫细胞化学显示形成集落的细胞白蛋白、转铁蛋白、角蛋白8和CD44呈阳性。克隆形成细胞表达白蛋白、转铁蛋白、α-1-抗胰蛋白酶、纤维蛋白原、谷氨酰胺合成酶、多种细胞色素P450和肝脏富集型转录因子(HNF3α、HNF4α、C/EBPα和C/EBPβ)。此外，细胞不仅表达肝脏分化功能基因，还表达肝干细胞标记物(Thy1.1、EpCAM、AFP)和SH标记物(CD44、D6.1A、BRI3)的基因。同时观察培养上清中白蛋白的分泌情况。我们的结果证明在成人肝脏中存在肝细胞前体细胞，并且这些细胞可以在透明质酸涂层培养皿的无血清培养液中生长。人类SHS可能是细胞移植以及药物和毒理学研究的有用来源。
Rat small hepatocytes (SHs) are committed progenitor cells that can differentiate into mature hepatocytes and can selectively proliferate in serum-free medium when they are cultured on hyaluronic acid (HA)-coated dishes. In this study we examined the separation of human SHs from adult human livers. We obtained liver tissues from the resected liver of 16 patients who underwent hepatic resections. Extracted liver specimens were clearly separate from the tumor regions with sufficient margins. Hepatic cells were isolated using the modified method of two-step collagenase perfusion. A low-speed centrifugation was performed and cells in the supernatant were finally cultured on HA-coated dishes in serum-free DMEM/F12 medium including nicotinamide, EGF, and HGF. Small-sized hepatocytes selectively proliferated to form colonies and many colonies continued growing for more than 3 weeks. The average number of cells in a colony was 38.6 ± 18.0, 79.0 ± 54.0, and 101.5 ± 115.7 at day 7, 14, and 21, respectively. About 0.04% of plated cells could form an SH colony. Immunocytochemistry showed that the cells forming a colony were positive for albumin, transferrin, keratin 8, and CD44. The results of RT-PCR showed that colony-forming cells expressed albumin, transferrin, α1-antitrypsin, fibrinogen, glutamine synthetase, many cytochrome P450s, and liver-enriched transcription factors (HNF3α, HNF4α, C/EBPα, and C/EBPβ). Furthermore, the cells expressed not only the genes of hepatic differentiated functions but also those of both hepatic stem cell marker (Thy1.1, EpCAM, AFP) and SH marker (CD44, D6.1A, BRI3). Albumin secretion into culture medium was also observed. Our results demonstrate the existence of hepatocyte progenitor cells in human adult livers, and the cells can grow in a serum-free medium on HA-coated dishes. Human SHs may be a useful source for cell transplantation as well as pharmaceutical and toxicological investigations.