title: Acyclic retinoid induces differentiation and apoptosis of murine hepatic stem cells.
abstract: BACKGROUND
  The therapeutic potential of acyclic retinoid (ACR), a synthetic retinoid, has been confirmed in experimental and clinical studies. Therapeutic targets include precancerous and cancer stem cells. As ACR is also involved in developmental processes, its effect on normal hepatic stem cells (HpSCs) should be investigated for understanding the underlying mechanisms. Here, we examined effects of the acyclic retinoid peretinoin on fresh isolated murine HpSCs.
  
  
  METHODS
  We isolated c-kit-CD29+CD49f+/lowCD45-Ter119- cells from murine fetal livers using flow cytometry. To evaluate the effect of ACR, we traced clonal expansion and analyzed cell differentiation as well as apoptosis during the induction process by immunofluorescent staining and marker gene expression.
  
  
  RESULTS
  ACR dose-dependently inhibited HpSCs expansion. Stem cell clonal expansion was markedly inhibited during the culture period. Moreover, ACR showed a significant promotion of HpSC differentiation and induction of cellular apoptosis. The expression of stem cell marker genes, Afp, Cd44, and Dlk, was downregulated, while that of mature hepatocyte genes, Alb and Tat, and apoptosis-related genes, Annexin V and Caspase-3, were upregulated. Flow cytometry showed that the proportion of Annexin V-positive cells increased after ACR incubation compared with the control. Data obtained by immunofluorescent staining for albumin and Caspase-3 corroborated the data on gene expression. Finally, we found that ACR directly regulates the expression of retinoic acid receptors and retinoid X receptors.
  
  
  CONCLUSIONS
  These findings indicate that ACR inhibits the clonal expansion of normal HpSCs in vitro and promotes the differentiation of immature cells by regulating receptors of retinoic acid.
authors.affiliation: Department of Regenerative Medicine, Graduate School of Medicine, Yokohama City University, Yokohama, Kanagawa, 236-0004, Japan. dillonguan277@gmail.com.
authors.forename: Hong-Bin
authors.initials: HB
authors.lastname: Guan
authors.affiliation: Department of Regenerative Medicine, Graduate School of Medicine, Yokohama City University, Yokohama, Kanagawa, 236-0004, Japan. nie.yunzhong@gmail.com.
authors.forename: Yun-Zhong
authors.initials: YZ
authors.lastname: Nie
authors.affiliation: Department of Regenerative Medicine, Graduate School of Medicine, Yokohama City University, Yokohama, Kanagawa, 236-0004, Japan. ywzheng@md.tsukuba.ac.jp.|Department of Advanced Gastroenterological Surgical Science and Technology, Faculty of Medicine, University of Tsukuba, Tsukuba, 305-8575, Japan. ywzheng@md.tsukuba.ac.jp.
authors.forename: Yun-Wen
authors.initials: YW
authors.lastname: Zheng
authors.affiliation: Department of Regenerative Medicine, Graduate School of Medicine, Yokohama City University, Yokohama, Kanagawa, 236-0004, Japan. abysszera@yahoo.co.jp.
authors.forename: Kazuya
authors.initials: K
authors.lastname: Takiguchi
authors.affiliation: Department of Histology and Embryology, Harbin Medical University, Harbin, 150081, China. ydhongwei@yahoo.com.cn.
authors.forename: Hong-Wei
authors.initials: HW
authors.lastname: Yu
authors.affiliation: Department of Regenerative Medicine, Graduate School of Medicine, Yokohama City University, Yokohama, Kanagawa, 236-0004, Japan. ranranzhang.0537@hotmail.com.
authors.forename: Ran-Ran
authors.initials: RR
authors.lastname: Zhang
authors.affiliation: Oregon Stem Cell Center, Oregon Health and Science University, Portland, OR, 97239, USA. libi@ohsu.edu.
authors.forename: Bin
authors.initials: B
authors.lastname: Li
authors.affiliation: Department of Regenerative Medicine, Graduate School of Medicine, Yokohama City University, Yokohama, Kanagawa, 236-0004, Japan. t126040f@yokohama-cu.ac.jp.
authors.forename: Tomonori
authors.initials: T
authors.lastname: Tsuchida
authors.affiliation: Department of Regenerative Medicine, Graduate School of Medicine, Yokohama City University, Yokohama, Kanagawa, 236-0004, Japan. zoukisai@yokohama-cu.ac.jp.|Advanced Medical Research Center, Yokohama City University, Yokohama, Kanagawa, 236-0004, Japan. zoukisai@yokohama-cu.ac.jp.
authors.forename: Hideki
authors.initials: H
authors.lastname: Taniguchi
mesh_terms: D000818:Animals; D017304:Annexin A5 / Q000096:biosynthesis; D017209:Apoptosis / Q000187:drug effects*; D002135:Calcium-Binding Proteins; D053148:Caspase 3 / Q000096:biosynthesis; D002454:Cell Differentiation / Q000187:drug effects*; D002478:Cells, Cultured; D015536:Down-Regulation; D005434:Flow Cytometry; D015696:Gene Products, tat / Q000096:biosynthesis; D018960:Hyaluronan Receptors / Q000096:biosynthesis; D036341:Intercellular Signaling Peptides and Proteins / Q000096:biosynthesis; D008099:Liver / Q000166:cytology*; D008115:Liver Regeneration / Q000502:physiology*; D051379:Mice; D008810:Mice, Inbred C57BL; D018168:Receptors, Retinoic Acid / Q000378:metabolism; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism; D014212:Tretinoin / Q000031:analogs & derivatives* / Q000494:pharmacology; D015854:Up-Regulation; D000509:alpha-Fetoproteins / Q000096:biosynthesis
chemical_list: D017304:Annexin A5; D002135:Calcium-Binding Proteins; C476399:Cd44 protein, mouse; C512925:Dlk1 protein, mouse; D015696:Gene Products, tat; D018960:Hyaluronan Receptors; D036341:Intercellular Signaling Peptides and Proteins; D018168:Receptors, Retinoic Acid; D000509:alpha-Fetoproteins; D014212:Tretinoin; C034534:3,7,11,15-tetramethyl-2,4,6,10,14-hexadecapentaenoic acid; D053148:Caspase 3