Abstract
Small hepatocyte-like progenitor cells (SHPCs) are known as liver stem/progenitor cells (LSPCs). SHPCs transiently appear and form clusters in rat livers treated with retrorsine (Ret) and a 70% partial hepatectomy (PH). The Ret/PH model has been used widely to analyze the effectiveness of cell transplantation and the mechanisms of LSPC proliferation. Laser microdissection (LMD) is a powerful tool that can excise and collect specific areas of cells from a tissue slice with a laser under a microscope. These cells exhibiting morphological alterations different from the surrounding cells may be analyzed by gene expression profiling. Specific markers of SHPCs have not yet been identified, in part, because it is difficult to isolate SHPCs from the liver using fluorescence or magnetic-activated cell sorting. To examine the underlying mechanism for SHPC growth, we established comprehensive gene expression profiles for SHPCs captured from liver sections using LMD. In this chapter, we introduce a method to isolate SHPCs from liver tissue sections using LMD for gene expression analysis.
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References
Forbes S, Vig P, Poulsom R et al (2002) Hepatic stem cells. J Pathol 197:510–518
Fausto N, Campbell JS (2003) The role of hepatocytes and oval cells in liver regeneration and repopulation. Mech Dev 120:117–130
Tanimizu N, Mitaka T (2014) Re-evaluation of liver stem/progenitor cells. Organogenesis 10:208–215
Alison MR, Islam S, Lim S (2009) Stem cells in liver regeneration, fibrosis and cancer: the good, the bad and the ugly. J Pathol 217:282–298
Chen YH, Chang MH, Chien CS et al (2013) Contribution of mature hepatocytes to small hepatocyte-like progenitor cells in retrorsine-exposed rats with chimeric livers. Hepatology 57:1215–1224
Petersen BE, Goff JP, Greenberger JS, Michalopoulos GK (1998) Hepatic oval cells express the hematopoietic stem cell marker Thy-1 in the rat. Hepatology 27:433–445
Mitaka T, Mikami M, Sattler GL et al (1992) Small cell colonies appear in the primary culture of adult rat hepatocytes in the presence of nicotinamide and epidermal growth factor. Hepatology 16:440–447
Mitaka T, Sato F, Mizuguchi T et al (1999) Reconstruction of hepatic organoid by rat small hepatocytes and hepatic nonparenchymal cells. Hepatology 29:111–125
Sugimoto S, Mitaka T, Ikeda S et al (2002) Morphological changes induced by extracellular matrix are correlated with maturation of rat small hepatocytes. J Cell Biochem 87:16–28
Tanimizu N, Ichinohe N, Yamamoto M et al (2017 Jan) Progressive induction of hepatocyte progenitor cells in chronically injured liver. Sci Rep 4(7):39990
Sasaki K, Kon J, Mizuguchi T et al (2008) Proliferation of hepatocyte progenitor cells isolated from adult human livers in serum-free medium. Cell Transplant 17:1221–1230
Kon J, Ooe H, Oshima H et al (2006) Expression of CD44 in rat hepatic progenitor cells. J Hepatol 45:90–98
Gordon GJ, Coleman WB, Hixson DC, Grisham JW (2000) Liver regeneration in rats with retrorsine-induced hepatocellular injury proceeds through a novel cellular response. Am J Pathol 156:607–619
Gordon GJ, Coleman WB, Grisham JW (2000) Temporal analysis of hepatocyte differentiation by small hepatocyte-like progenitor cells during liver regeneration in retrorsineexposed rats. Am J Pathol 157:771–786
Laconi E, Oren R, Mukhopadhyay DK et al (1998) Long-term, near-total liver replacement by transplantation of isolated hepatocytes in rats treated with retrorsine. Am J Pathol 153:319–329
Ichinohe N, Kon J, Sasaki K et al (2012) Growth ability and repopulation efficiency of transplanted hepatic stem cells, progenitor cells, and mature hepatocytes in retrorsine-treated rat livers. Cell Transplant 21:11–22
Ichinohe N, Ishii M, Tanimizu N et al (2017) Transplantation of Thy1+ cells accelerates liver regeneration by enhancing the growth of small hepatocyte-like progenitor cells via IL17RB Signaling. Stem Cells 35:920–931
Ichinohe N, Ishii M, Tanimizu N et al (2021) Extracellular vesicles containing miR-146a-5p secreted by bone marrow mesenchymal cells activate hepatocytic progenitors in regenerating rat livers. Stem Cell Res Ther 12:312
Aguilar-Bravo B, Sancho-Bru P (2019) Laser capture microdissection: techniques and applications in liver diseases. Hepatol Int 13:138–147
Vandewoestyne M, Goossens K, Burvenich C et al (2013) Laser capture microdissection: should an ultra- violet or infrared laser be used? Anal Biochem 439:88–98
Gallagher RI, Blakely SR, Liotta LA, Espina V (2012) Laser capture microdissection: ArcturusXT infrared capture and UV cutting methods. Methods in Molecular Biology. Humana Press, New York, pp 157–178
Espina V, Wulfkuhle JD, Calvert VS et al (2006) Laser-capture microdissection. Nat Protoc 1:586–603
Liu H, Mcdowell TL, Hanson NE et al (2014) Laser capture microdissection for the investigative pathologist. Vet Pathol 51:257–269
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Ichinohe, N., Tanimizu, N., Mitaka, T. (2022). Isolation of Small Hepatocyte-Like Progenitor Cells from Retrorsine/Partial Hepatectomy Rat Livers by Laser Microdissection. In: Tanimizu, N. (eds) Hepatocytes. Methods in Molecular Biology, vol 2544. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2557-6_13
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DOI: https://doi.org/10.1007/978-1-0716-2557-6_13
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