Human placental hydrolysate promotes the long-term culture of hepatocyte-like cells derived from canine bone marrow

Abstract

Long-term culture of canine artificial hepatocytes has not been established. We hypothesized that human placental hydrolysate (hPH) may support the long-term culture of differentiated hepatocyte-like cells. Canine bone marrow cells were cultured using modified hepatocyte growth medium supplemented with hPH. Quantitative reverse transcription polymerase chain reaction (RT-PCR) and immunocytochemical analysis for albumin, qualitative RT-PCR for cytochrome P450 1A1 (CYP1A1), hepatocyte growth factor (HGF), Cytokeratin 7 (CK7), CD90, CD44, and CD34, and functional analyses of CYP450 activity and low-density lipoprotein (LDL) uptake were performed. Cultured hepatocyte-like cells were able to maintain hepatocyte characteristics, including morphology, albumin synthesis, CYP450 activity, and LDL uptake for 80 days. Thus, hPH may be a potential facilitator for the long-term culture of hepatocyte-like cells. Clinicopathologically, this culture protocol of artificial hepatocytes will contribute to liver function evaluation.

Keywords: bone marrow; dog; hepatocyte; long-term culture; placenta.

Fig. 1.

The morphology of cultured canine bone marrow cells (cBMCs) on days 7 (A), 21 (B), 60 (C), and 80 (D). cBMCs attached to the bottom of the flask were spindle-like shaped on day 7, and became polygonal (pentagonal), adult hepatocyte-like cells, on days 21, 60, and 80. Scale bars represent 20 µm.

Fig. 2.

F-actin staining and immunocytochemical staining of albumin in cultured bone marrow (BM) cells. (A) F-actin staining of cultured BM on day 21, viewed using a confocal microscope. Strong fluorescence was observed within the cell membrane, showing epithelial cell characteristics. Scale bar represents 20 µm. (B) Immunofluorescent staining of BM cells with anti-human albumin polyclonal antibody. Positively stained cells on day 80 (C), which were more strongly stained compared to cells in the negative control (B), were counted within ten randomly selected areas; 10–15% of hepatocyte-like cells were positive for albumin. Scale bars represent 50 µm.

Fig. 3.

Gene expression analysis of cultured canine bone marrow cells using qualitative RT-PCR. (A) Qualitative RT-PCR analysis revealed that albumin (ALB) and cytochrome P450 (CYP1A1) were expressed in cultured bone marrow cells on days 21 and 80. Hepatocyte growth factor (HGF), CD90, and CD44 were detected in bone marrow cells and in cultured bone marrow cells on days 21 and 80. CD34 was only detected in bone marrow cells. Hepatocytes from adult canine liver expressed ALB, HGF, CYP1A1, and CK7. (B) Quantitative RT-PCR analysis of albumin mRNA. Using the 2^-ΔΔCt method, the mRNA level of hepatocyte-like cells on day 14 was set to 1 and the relative mRNA levels on days 21, 28, 35, and 80 were expressed as mean ± standard error of the mean. * (P<0.05) indicates a significant difference from the bone marrow cells from the same dogs.

Fig. 4.

In vitro functional characterization of hepatocyte-like cells differentiated from bone marrow cells. Nuclei were stained using 4′,6-diamidino-2-phenylindole (blue), and the photomicrographs were overlaid with those of cytochrome P450 (CYP450) and the low-density lipoprotein (LDL) fluorescence. CYP450 and LDL were detected in differentiated cells at day 80 (B, D). N.C. indicates negative control, differentiated cells without CYP450 and LDL fluorescence (A, C). Scale bars represent 50 µm.