| The liver is an important organ in the body where various metabolisms of exogenous and endogenous substrates are performed. Normal liver cell turnover and the restore of liver structure and function after partial hepatectomy was mainly due to mitosis of mature hepatocytes, which was verified mainly by studies performed on animals. However, when the liver was severely damaged by physical or chemical factors, it was the hepatic stem cells that restored the liver by proliferation and differentiation, the hepatic stem cells were generally recognized as those liver progenitor cells that can proliferate intensively and differentiate into mature hepatocytes and bile duct cells. It had been shown that isolation and culture of hepatic stem cells and further studies on it were valuable for understanding liver development, liver tumor and liver regeneration. At present, studies on hepatic stem cells were still on their initial stage to isolate, culture, and establish cell lines, which mainly performed on rodent animals. As to studies on human hepatic stem cells, Malhi H et al (2002) had reported liver epithelial progenitor cell lines from fetal human liver. Without primary fractionation and clonal subculture, the cell lines showed a mixture phenotype and spontaneously differentiation. Based on former successful isolation and culture of hepatic stem cells from the mice, we tried to establish long-term stable culture of human hepatic stem cell lines from early human fetus, which would provide a ideal model for researching medical and biological topics such as liver tumor and liver regeneration, as well as provide abundant cell source and related knowledge for studies on cell therapy for liver metabolic diseases, including liver failure caused by various factors.We isolated putative stem cells from aborted human fetus in 4.5~6th week of gestation (because it was known that the hepatic diverticulum arised at the 4th week of gestation, and that fetal livers during this stage consisted higher percentage of stem cells) . By optimization the culture medium and repeatedclonal growth we finally established 11 cell lines that could be continuously and stably cultured in vitro. With similar cell shape, proliferation ability and gene expression profile, these cell lines were named human fetal liver derived stem cells (hFLSC). We mainly characterized one of the hFLSC cell lines as follows:(1) hFLSC had a homogenous small triangle shape. The hFLSC cell is small in size, with a round nuclear and 1~3 nucleoli. processes were seen between adjacent cells. Subcultured cells maintained the similar shape. Ultrastructure study observed a high cytoplasm to nuclear ratio and few organelles in the cytoplasm.(2) The hFLSC cell had powerful proliferation capability. The cell line had been stably propagated in culture up to 25 passages over 7 months, it had been demonstrated that they had a normal karyotype, and no tumors formed four months after they were injected subcutaneously into SCID mice.(3) The hFLSC cell expressed various kinds of markers for different lineage cells. RT-PCR, immunocytochemistry and fluorescence-activated cell sorting(FACS) were used to detect their gene expression profile. And the results showed that they not only expressed hepatic stem cell related markers(such as AFP, ALB, CK8, CK18, CK19, GGT, DPPIV, c-Met, Thy-1 and c-kit), but also expressed some mesenchymal cell markers(such as CD44, CD29, and SDF-1). while the expression of hematopoeitic markers(CD34 and CD45) were not detected. Since the expression of CD44 and CD29 was more than 99% by flow cytometric analysis, and the expression of AFP, ALB, CK8, CK18, GGT, DPPIV, c-Met and c-kit were detected by immunocytochemistry in almost all of the cells, we supposed that there was coexpression of mesenchymal markers and hepatic stem cell related markers in hFLSC. Moreover, the expression of stem cell transcription factor Oct-4 was detected by RT-PCR. The result suggests that hFLSC may be a primitive stem cell in the early human fetal liver.(4) in... |
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