Gastroenterology

Gastroenterology

Volume 137, Issue 3, September 2009, Pages 1114-1126.e14
Gastroenterology

Basic—Liver, Pancreas, and Biliary Tract
Enrichment and Clonal Culture of Progenitor Cells During Mouse Postnatal Liver Development in Mice

https://doi.org/10.1053/j.gastro.2009.06.001Get rights and content

Background & Aims

Stem and progenitor cells exist in normal postnatal livers. However, it has not been possible to clonally isolate or analyze postnatal liver stem/progenitor-like cells (PLSCs) derived from noninjured livers because of a lack of specific surface markers. This study aimed to establish a primary culture system for clone-sorted PLSCs.

Methods

To investigate proliferation and differentiation of PLSCs, subpopulations of nonparenchymal cells derived from noninjured livers were purified and cultured using a single-cell culture system. Cells were grown in fetal liver cell-derived conditioned medium in the presence of the Rho-associated kinase (ROCK) inhibitor Y-27632.

Results

We identified CD13 and CD133 as markers expressed on the PLSC-containing population in noninjured livers and established an efficient single-cell culture system to clonally analyze PLSCs. Culture of PLSCs is difficult, even using conditioned medium, but the addition of Y-27632 increased PLSC cell proliferation. The proportion of progenitor cells among nonparenchymal cells decreased during postnatal liver development; however, a PLSC population was still preserved in 3-month-old mice. Long-term cultivated cells derived from clone-sorted cells in normal livers were established and were called normal-liver-derived stem-like cells (NLS cells). NLS cells could differentiate into hepatocyte-like and cholangiocyte-like cells under appropriate culture conditions and underwent self-renewal-like activity in serial reclone-sorted culture. CD13 and CD133 were expressed on progenitor cells derived from fetal and postnatal liver, whereas CD49f (integrin α6 subunit) was strongly expressed only on PLSCs.

Conclusions

These results demonstrate the presence of progenitor cells in the CD13+CD49f+CD133+ subpopulation of nonhematopoietic cells derived from noninjured postnatal livers.

Section snippets

Cell Preparation From Postnatal Livers

Hepatocytes and nonparenchymal cells were isolated from postnatal livers following a 2-step collagenase digestion.26 Perfused liver tissues were subsequently dissociated with 0.05% collagenase solution in 10 minutes at 37°C. The mature hepatocyte fraction was separated from nonparenchymal cells by several episodes of low-speed centrifugation (50g for 1 minute). Nonparenchymal cells were collected by centrifugation (1200 rpm for 5 minutes). Dead cell debris was removed by centrifugation in 25%

Progenitor Cells Derived From CD45Ter119c-KitSca1 Population of Normal Postnatal Livers

Treatment with 3,5-diethoxycarbonyl-1,4-dihydrocollidine induces proliferation of oval cells, candidate bipotent progenitor cells, in mouse postnatal livers (“injured livers”).31, 32, 33 The phenotypes of stem/progenitor cells in normal (naive, noninjured) livers remain unknown. To identify the specific surface molecules of PLSCs, we used 2- to 3-week-old mice because preliminary experiments revealed that cells derived from young mice were highly able to form colonies (data not shown).

Discussion

In this study, we established a new primary culture system for clone-sorted cells derived from normal postnatal mouse livers. As shown in Figure 7, we speculate that stem/progenitor cell fraction phenotypes change during liver ontogeny. CD13 and CD133 were common to fetal hepatoblasts and PLSCs, whereas CD49f was detected only on PLSCs. Fetal CD45Ter119c-KitCD13+CD49f−/lowCD133+ cells and postnatal CD45Ter119c-KitSca1CD13+CD49f+CD133+ cells have similar capacities to form large colonies

References (41)

  • B.E. Petersen et al.

    Mouse A6-positive hepatic oval cells also express several hematopoietic stem cell markers

    Hepatology

    (2003)
  • S. Kakinuma et al.

    Analyses of cell surface molecules on hepatic stem/progenitor cells in mouse fetal liver

    J Hepatol

    (2009)
  • S. Paku et al.

    Origin and structural evolution of the early proliferating oval cells in rat liver

    Am J Pathol

    (2001)
  • M. Perez-Moreno et al.

    Sticky business: orchestrating cellular signals at adherens junctions

    Cell

    (2003)
  • H. Yao et al.

    RhoA promotes differentiation of WB-F344 cells into the biliary lineage

    Differentiation

    (2009)
  • M. Mimeault et al.

    Concise review: recent advances on the significance of stem cells in tissue regeneration and cancer therapies

    Stem Cells

    (2006)
  • N. Fausto et al.

    Liver regeneration

    Hepatology

    (2006)
  • T. Cantz et al.

    Stem cells in liver regeneration and therapy

    Cell Tissue Res

    (2008)
  • N. Tanimizu et al.

    Isolation of hepatoblasts based on the expression of Dlk/Pref-1

    J Cell Sci

    (2003)
  • A. Suzuki et al.

    Clonal identification and characterization of self-renewing pluripotent stem cells in the developing liver

    J Cell Biol

    (2002)
  • Cited by (88)

    • The stem cells in liver cancers and the controversies

      2018, Stem Cells and Cancer in Hepatology: From the Essentials to Application
    • Plasticity of liver epithelial cells in healthy and injured livers

      2018, Stem Cells and Cancer in Hepatology: From the Essentials to Application
    • The role of stem cells in the hepatobiliary system and in cancer development: A surgeon’s perspective

      2018, Stem Cells and Cancer in Hepatology: From the Essentials to Application
    View all citing articles on Scopus

    Conflicts of interest The authors disclose no conflicts.

    Funding Supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology in Japan, Grants-in-Aid from the Japan KAMPO Medicine Manufacturers Association, and the Kampou Science Foundation.

    View full text