Biliary epithelial cell differentiation from embryonic stem cells in vitro

An Bin Hu, Xiaoshun He, Ji Ye Cai, Qi Chang Zheng

Research output: Contribution to journalArticle

2 Scopus citations

Abstract

Background: Previous research demonstrated that embryonic stem (ES) cells can be induced to differentiate into hepatocytes. It is feasible to induce ES cells to differentiate into biliary epithelial (BE) cells by specific induction conditions based on the previous research. Objective: To validate the feasibility to induce ES cells to differentiate into BE cells in the presence of cell growth factors. Design: Single sample observation. Setting: First Hospital of Sun Yat-sen University. Materials: Undifferentiated BALB/C-ES cell line was provided by the Experimental Animal Center of Sun Yat-sen University; Transforming growth factor, acidic fibroblast growth factor, hepatocyte growth factor, epidermal growth factor, and keratinocyte growth factor by Sigma, USA. First antibody: anti-mouse cytokeratin 7 (CK7) and CK19 by DAKO, Denmark; indirect immunofluorescence from Biodesign, USA; and inverted contrast fluorescence microscope by OLYMPUS 1X70-S8F2, Japan. Methods: The experiment was performed at Central Laboratory of the First Affiliated Hospital of Sun Yat-sen University from October 2004 to June 2005. During the culture of embryonic bodies (EBs) derived from ES cells, some growth factors such as Transforming growth factor, acidic fibroblast growth factor, hepatocyte growth factor and epidermal growth factor were respectively added into medium to induce BE cells differentiation. ES cells in culture condition with no added growth factors served as control. The differentiation status and three-dimensional duct-like structure formation of ES cells was observed dynamically by inverted microscope. The markers of BE cells such as CK7, CK19 and gamma-glutamyltransferase (GGT) were detected by immunocytochemistry and GGT-positive cells were observed. Main outcome measures: 1 Cell growth and three-dimensional duct-like structure formation; 2 expressions of the marker proteins of BE cells such as CK7 and CK19; 3 expression of GGT, the marker enzyme of BE cells, and GGT-positive cell morphology. Results: Small EBs formed and floated in culture suspension after 10 hours of ES cell culture. Many three-dimensional duct-like structures appeared in EBs culture system within 10 differentiation days. Cells were arranged in cocentric circle and lamellar shape; the inner layer cells aligned tightly but became sparse gradually. The cells had best viability on day 20, and disintegrated in strands in culture solution on day 36. Duct-like structure was found on day 13 in control group and disintegrated on day 27. CK7 was expressed on the day of duct-like structure formation, and CK19 expression was found on day 13. Both expressions were elevated with culture time thereafter. CK7 and CK19 were expressed in control group on days 13 and 15, respectively. GGT expressed in duct-like structures after growth factors were added, indicating there was BE cells in these structures. At early stage of duct-like structure formation, cells connected too closely to identify single cell structure, but cell structure became clear in further culture. GGT-positive cells had morphological characters consistent with mouse normal BE cells such as: the cells were polygonal or quadrate shapes; the nucleus was big and round and situated at the center of cells; the organelles were lacking in cytoplasm. Conclusion: ES cells can differentiate into BE cells in presence of certain growth factors and form bile duct-like structures.

Original languageEnglish (US)
Pages (from-to)5783-5787
Number of pages5
JournalJournal of Clinical Rehabilitative Tissue Engineering Research
Volume12
Issue number29
StatePublished - Jul 15 2008

ASJC Scopus subject areas

  • Orthopedics and Sports Medicine
  • Biomedical Engineering
  • Clinical Biochemistry

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