Development of a chemically defined medium and discovery of new mitogenic growth factors for mouse hepatocytes: Mitogenic effects of FGF1/2 and PDGF

Chemically defined serum-free media for rat hepatocytes have been useful in identifying EGFR ligands and HGF/MET signaling as direct mitogenic factors for rat hepatocytes. The absence of such media for mouse hepatocytes has prevented screening for discovery of such mitogens for mouse hepatocytes. We present results obtained by designing such a chemically defined medium for mouse hepatocytes and demonstrate that in addition to EGFR ligands and HGF, the growth factors FGF1 and FGF2 are also important mitogenic factors for mouse hepatocytes. Smaller mitogenic response was also noticed for PDGF AB. Mouse hepatocytes are more likely to enter into spontaneous proliferation in primary culture due to activation of cell cycle pathways resulting from collagenase perfusion. These results demonstrate unanticipated fundamental differences in growth biology of hepatocytes between the two rodent species. Copyright: © 2014 Reekie et al

High fidelity copy number analysis of formalin-fixed and paraffin-embedded tissues using affymetrix cytoscan HD chip

Detection of human genome copy number variation (CNV) is one of the most important analyses in diagnosing human malignancies. Genome CNV detection in formalin-fixed and paraffin-embedded (FFPE) tissues remains challenging due to suboptimal DNA quality and failure to use appropriate baseline controls for such tissues. Here, we report a modified method in analyzing CNV in FFPE tissues using microarray with Affymetrix Cytoscan HD chips. Gel purification was applied to select DNA with good quality and data of fresh frozen and FFPE tissues from healthy individuals were included as baseline controls in our data analysis. Our analysis showed a 91% overlap between CNV detection by microarray with FFPE tissues and chromosomal abnormality detection by karyotyping with fresh tissues on 8 cases of lymphoma samples. The CNV overlap between matched frozen and FFPE tissues reached 93.8%. When the analyses were restricted to regions containing genes, 87.1% concordance between FFPE and fresh frozen tissues was found. The analysis was further validated by Fluorescence In Situ Hybridization on these samples using probes specific for BRAF and CITED2. The results suggested that the modified method using Affymetrix Cytoscan HD chip gave rise to a significant improvement over most of the previous methods in terms of accuracy in detecting CNV in FFPE tissues. This FFPE microarray methodology may hold promise for broad application of CNV analysis on clinical samples. © 2014 Yu et al

Interactions between FGF2 and PDGF AA and BB in stimulation of DNA synthesis in mouse hepatocyte cultures.

<p>Y-axis indicates percent of labeled nuclei due to incorporation of bromodeoxyuridine into DNA. The growth factors added to the medium are listed under the X-axis.</p

Mouse hepatocyte proliferation in the final formulation of the MHGM medium.

<p>Y-axis indicates percent of labeled nuclei due to incorporation of bromodeoxyuridine into DNA. The growth factors added to the medium are listed under the X-axis. CTR: Control cultures, with no growth factors added. Growth factors were added at 24 hours after plating of hepatocytes. D 0–2 (etc.) indicates days in culture after addition of the growth factors. (For growth factor concentration, please see Materials and Methods).</p

Rat hepatocyte proliferation in the MHGM medium.

<p>Y-axis indicates percent of labeled nuclei due to incorporation of bromodeoxyuridine into DNA. The scale of Y-axis is kept identical to that of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095487#pone-0095487-g004" target="_blank">figure 4</a>, for direct comparison of the responses of mouse and rat hepatocytes. The growth factors added to the medium are listed under the X-axis. CTR: Control cultures, with no growth factors added. Growth factors were added at 24 hours after plating of hepatocytes. D 0–2 (etc.) indicates days in culture after addition of the growth factors. (For growth factor concentration, please see Materials and Methods).</p

Intense vacuolation of hepatocyte cytoplasm in a preliminary formulation of the mouse hepatocyte growth medium (MHGM).

<p>Elimination of nicotinamide removed this cytoplasmic change. A: Phase contrast photomicrograph of the hepatocytes in culture showing intense valuation of the cytoplasm (Magnification: 100X). B: Electron micrograph (5,000X) of hepatocytes carrying vacuoles. There is no apparent connection with bile canaliculi or autophagosomes. C: Oil Red-O stain for lipid demonstrates that the vacuoles observed did not contain lipids.</p

Suppression of spontaneous proliferation of mouse hepatocytes in the presence of 10

<p>^−<b>6</b><b> M dexamethasone.</b> Tritiated thymidine was added continually in the cultures, from 4 hours after cell plating following perfusion of the liver by collagenase. Cultures were harvested at the indicated time points in the X-axis. Addition of HGF and EGF is indicated as “GFs”. (For growth factor concentration, please see Materials and Methods).</p

Proliferation of mouse hepatocytes maintained in the rat hepatocyte (HGM) medium.

<p>Y-axis indicates incorporation of tritiated thymidine per µg DNA. The growth factors added to the medium are listed under the X-axis. CTR: Control cultures, with no growth factors added. Growth factors were added at 24 hours after plating of hepatocytes. D 0–2 (etc.) indicates days in culture after addition of the growth factors. (For growth factor concentration, please see Materials and Methods).</p