Establishment of an albumin and cytokeratin 19 genetically-modified embryonic stem cell line and evaluation of its hepatoblast differentiation capacities / 中华肝脏病杂志

ABSTRACT

<p><b>OBJECTIVE</b>To establish a gene-modified embryonic stem (ES; E14.1-2) cell line with hepatoblast differentiation reporter genes, albumin (ALB) and cytokeratin 19 (CK19), labeled to facilitate study of their potential applicability as differentiated hepatoblasts.</p><p><b>METHODS</b>Two expression vectors were constructed, one with the ALB promotor driving the enhanced green fluorescent protein (EGFP) and anti-neomycin genes (pAlb-EGFP), and the other with the CK19 promotor driving the red fluorescence protein and anti-hygromycin genes (pCK19-hCD25-IRES-tdTOMATO). The linearized vectors were electroporated into the E14.1 line, and double reporter genes-modified ES cells (E14.1-2) were selected by neomycin and hygromycin. E14.1-2 hepatoblast differentiation was induced by exposure to growth factors (BMP4 and bFGF) and evidenced by embryoid body formation. Fluorescence-activated cell sorting (FACS) and reverse transcription-polymerase chain reaction (RT-PCR) were used to confirm whether differentiated cells were hepatoblast-like and to quantify the differentiation efficiency.</p><p><b>RESULTS</b>The pAlb-EGFP and pCK19-hCD25-IRES-tdTOMATO vectors were shown to specifically activate ALB and CK19 expression. The E14.1-2 cell line with labeled ALB and CK19 was established, and shown to have pluripotency by RT-PCR detection of pluripotent markers' expression, namely Oct4 and SSEA-1. After 22 days of induction, 21.27% of the differentiated hepatoblasts were detected by FACS as positive for ALB and CK19 expression.</p><p><b>CONCLUSIONS</b>A gene-modified ES cell line was generated with hepatocyte differentiation reporter genes ALB and CK19 labeled. The differentiation of the resultant E14.1-2 line was technically simple to qualify and quantify, and will likely aid future studies of hepatoblast characteristics.</p>