Histochem Cell Biol (2011) 135:581–591 cells isolated from DPPIV? normal liver donor (groups B). Cells were isolated as described above and were delivered via portal vein infusion, suspended in 0.3 ml of Williams E medium. Animals from the two groups were killed 1 month after transplantation (Tx); livers were excised, cut into 1–2 mm thick slices and grossly examined for the presence of hepatic nodules and/or any other visible lesions. Liver samples taken from each liver were either ﬁxed in 10% buffered formaldehyde for standard histological analysis (H&E staining) or snap frozen. Histochemical determina- tion of DPPIV enzyme activity was performed as described (Laconi et al. 2001b). Immunohistochemistry for Ki67, Cx32, and E-cadherin was performed using speciﬁc monoclonal antibodies (anti- Ki67 from Abcam, cat # 16667; anti-Cx32 from Zymed Labs, cat # 34-5700, anti-E-cadherin from Santa Cruz, cat. # sc-8426). Detection of primary antibodies was accom- plished using the avidin/biotin peroxidase system (Vecta- stain ABC kit; Vector Laboratories, Burlingame, CA). Colocalization of DPPIV with Cx32 and MMP-2 Tissues were immunostained for the ﬁrst antigen [anti- Cx32 (1:5,000) and anti-MMP-2 (1:100)] (Neomarkers Asbach, Germany) using Alexa 488 conjugated goat anti- rabbit or anti-mouse IgG (Molecular Probes Goettingen, Germany) for ﬂuorescence detection (1:400, 1 h at RT) and then further processed with the second immunostaining protocol. After rehydration in Tris buffer, specimens were blocked and subsequently incubated with the second pri- mary antibody (anti-DPPIV, 1:100), rinsed with Tris buffer and exposed to the second Alexa Fluor 568 goat anti- mouse IgG2a (Molecular Probes Goettingen, Germany) (1:400, 1 h at RT). Slides were ﬁnally covered with Vec- tashield mounting medium with DAPI (1 ll/ml) (Vector Laboratories, UK) to visualise cell nuclei. Negative con- trols were carried out for each antibody by omitting the primary antibody from the protocol. Multiple immuno- ﬂuorescence-labelled specimens were serially excited and observed with the TEXAS Red-, FITC- and UV-ﬁlter set on an inverted confocal microscope (LEICA DM IRE2, Bensheim, Germany). Pictures of each ﬁlter set were dig- itally merged using layering technology software (Leica FW 4000, Version 1.1). RNA isolation and RT-PCR Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. RNA integrity and purity were conﬁrmed by 1% agarose gel electrophoresis and OD260/OD280 nm absorption ratio 583 [1.8. Two grams of DNase-I treated RNA of each sample were reverse-transcribed using Promega reagents (Pro- mega, Madison, WI). The resulting cDNA was analysed by PCR using the following primers and conditions: b2-microglobulin (FWD: 50-GTCGTGCTTGCCATTC AGA-30; REV: 50-TGAAGAAGATGGTGTGCTCA-30; Ta: 48 C; 30 cycles); MMP-2 (FWD: 50-TCCAGGAGCTCT ATGGGCCCTC-30; REV: 50- GCAGCCCAGCCAGTCC GATT-30; Ta: 63 C; 40 cycles). PCR thermal proﬁle was as follows: 95 C for 2 min, then 95 C for 30 s, Ta for 1 min, 72 C for 1 min for an appropriate number of cycles, fol- lowed by extension at 72 C for 5 min. PCR products were visualized by 2% agarose gel electrophoresis. Results The growth pattern of normal and nodular hepatocytes Normal transplanted hepatocytes integrated well in the recipient liver, forming clusters of irregular shape which were macroscopically indistinguishable in the context of the host tissue. No signs of compression of the sur- rounding tissue were evident during the growth of normal cells derived from donors. The distribution of DPPIV enzyme activity displayed the typical chicken-wire pat- tern, consistent with its normal bile canalicular location (Fig. 1a, c). However, the growth pattern of transplanted nodular hepatocytes was remarkably different. These cells formed focal lesions that were readily identiﬁable as greyish- brown areas on macroscopic examination, ranging in size from \1 to 3 mm in diameter, both on the liver surface and on sectioning. These lesions were mostly round or oval in shape and were sharply demarcated from the surrounding host tissue (Fig. 1b, d). The latter displayed signs of compression, as indicated by the presence of concentric hepatocyte plates distributed around larger nodules (Fig. 1f). Moreover, histochemical detection of DPPIV conﬁrmed the altered polarity of nodular hepatocytes, with no deﬁned pattern in the protein enzyme distribution (Fig. 1b, d). Many cells in nodules expressed cytoplasmic DPPIV activity with relatively scarce membrane localiza- tion, which became even more evident on immunoﬂuo- rescence examination (Fig. 1d). The surface receptor CD44, which is normally found in non-parenchymal liver cells and is up-regulated in different types of cancers, including hepatocellular carcinoma (Yang et al. 2010), was not expressed by nodular hepatocyte clusters derived from transplanted cells (Fig. 1g, h). How- ever, scattered CD44-positive cells were present both inside nodules and in surrounding liver exposed to RS (Fig. 1h), as already reported in the literature (Kon et al. 2009). 123