title: A Convenient and Efficient Method to Enrich and Maintain Highly Proliferative Human Fetal Liver Stem Cells.
abstract: Pluripotent human hepatic stem cells have broad research and clinical applications, which are, however, restricted by both limited resources and technical difficulties with respect to isolation of stem cells from the adult or fetal liver. In this study, we developed a convenient and efficient method involving a two-step in situ collagenase perfusion, gravity sedimentation, and Percoll density gradient centrifugation to enrich and maintain highly proliferative human fetal liver stem cells (hFLSCs). Using this method, the isolated hFLSCs entered into the exponential growth phase within 10 days and maintained sufficient proliferative activity to permit subculture for at least 20 passages without differentiation. Immunocytochemistry, immunofluorescence, and flow cytometry results showed that these cells expressed stem cell markers, such as c-kit, CD44, epithelial cell adhesion molecule (EpCAM), oval cell marker-6 (OV-6), epithelial marker cytokeratin 18 (CK18), biliary ductal marker CK19, and alpha-fetoprotein (AFP). Gene expression analysis showed that these cells had stable mRNA expression of c-Kit, EpCAM, neural cell adhesion molecule (NCAM), CK19, CK18, AFP, and claudin 3 (CLDN-3) throughout each passage while maintaining low levels of ALB, but with complete absence of cytochrome P450 3A4 (C3A4), phosphoenolpyruvate carboxykinase (PEPCK), telomeric repeat binding factor (TRF), and connexin 26 (CX26) expression. When grown in appropriate medium, these isolated liver stem cells could differentiate into hepatocytes, cholangiocytes, osteoblasts, adipocytes, or endothelial cells. Thus, we have demonstrated a more economical and efficient method to isolate hFLSCs than magnetic-activated cell sorting (MACS). This novel approach may provide an excellent tool to isolate highly proliferative hFLSCs for tissue engineering and regenerative therapies.
authors.affiliation: 1 Institute of Health and Environmental Medicine , Tianjin, China .|2 Key Laboratory of Risk Assessment and Control for Environment & Food Safety , Tianjin, China .
authors.forename: Xuan
authors.initials: X
authors.lastname: Guo
authors.affiliation: 1 Institute of Health and Environmental Medicine , Tianjin, China .|2 Key Laboratory of Risk Assessment and Control for Environment & Food Safety , Tianjin, China .
authors.forename: Shu
authors.initials: S
authors.lastname: Wang
authors.affiliation: 3 Peking Union Medical College Hospital , Chinese Medical Academy, Beijing, China .
authors.forename: Ya-ling
authors.initials: YL
authors.lastname: Dou
authors.affiliation: 1 Institute of Health and Environmental Medicine , Tianjin, China .|2 Key Laboratory of Risk Assessment and Control for Environment & Food Safety , Tianjin, China .
authors.forename: Xiang-fei
authors.initials: XF
authors.lastname: Guo
authors.affiliation: 1 Institute of Health and Environmental Medicine , Tianjin, China .|2 Key Laboratory of Risk Assessment and Control for Environment & Food Safety , Tianjin, China .
authors.forename: Zhao-li
authors.initials: ZL
authors.lastname: Chen
authors.affiliation: 1 Institute of Health and Environmental Medicine , Tianjin, China .|2 Key Laboratory of Risk Assessment and Control for Environment & Food Safety , Tianjin, China .
authors.forename: Xin-wei
authors.initials: XW
authors.lastname: Wang
authors.affiliation: 1 Institute of Health and Environmental Medicine , Tianjin, China .|2 Key Laboratory of Risk Assessment and Control for Environment & Food Safety , Tianjin, China .
authors.forename: Zhi-qiang
authors.initials: ZQ
authors.lastname: Shen
authors.affiliation: 1 Institute of Health and Environmental Medicine , Tianjin, China .|2 Key Laboratory of Risk Assessment and Control for Environment & Food Safety , Tianjin, China .
authors.forename: Zhi-gang
authors.initials: ZG
authors.lastname: Qiu
authors.affiliation: 1 Institute of Health and Environmental Medicine , Tianjin, China .|2 Key Laboratory of Risk Assessment and Control for Environment & Food Safety , Tianjin, China .
authors.forename: Min
authors.initials: M
authors.lastname: Jin
authors.affiliation: 1 Institute of Health and Environmental Medicine , Tianjin, China .|2 Key Laboratory of Risk Assessment and Control for Environment & Food Safety , Tianjin, China .
authors.forename: Jun-wen
authors.initials: JW
authors.lastname: Li
mesh_terms: D000328:Adult; D015415:Biomarkers / Q000378:metabolism*; D015815:Cell Adhesion Molecules / Q000378:metabolism; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D049109:Cell Proliferation; D002478:Cells, Cultured; D000072259:Connexin 26; D017630:Connexins; D005260:Female; D053686:Fetal Stem Cells / Q000166:cytology* / Q000378:metabolism; D005333:Fetus / Q000166:cytology* / Q000378:metabolism; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D020869:Gene Expression Profiling; D022781:Hepatocytes / Q000166:cytology* / Q000378:metabolism; D006801:Humans; D007124:Immunoenzyme Techniques; D016130:Immunophenotyping; D008099:Liver / Q000166:cytology* / Q000378:metabolism; D010641:Phenotype; D011247:Pregnancy; D011262:Pregnancy Trimester, Second; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D015415:Biomarkers; D015815:Cell Adhesion Molecules; D017630:Connexins; C000607042:GJB2 protein, human; D012333:RNA, Messenger; D000072259:Connexin 26