1. Volume 39, Issue 1, Pages 17-23 (July 2003)
Sera from liver failure patients and a demethylating agent stimulate transdifferentiation of murine bone marrow cells into hepatocytes in coculture with nonparenchymal liver cells 
Shintaro Yamazaki, Kenji Miki, Kiyoshi Hasegawa, Masataka Sata, Tadatoshi Takayama, Masatoshi Makuuchi 
Journal of Hepatology 
Volume 39, Issue 1, Pages (July 2003)
DOI: /S (03)

2. Fig. 1
Protocol for coculture of BMCs and NPCs. Liver NPCs from wild-type mice were cultured for 2 days in basal medium. BMCs from GFP transgenic mice were pretreated with AZA (5μmol/ml) for 12 h and added to the NPCs. HGF (50 ng/ml), OSM (20 ng/ml), DEX (10−7M) and HSLF (5%) were added to the basal medium. FACS sorting, immunocytochemistry and RT–PCR were performed after 14 days of coculture.
Journal of Hepatology  , 17-23DOI: ( /S (03) )

3. Fig. 2
Morphological changes in BMCs cocultured with NPCs. A BMC-derived hepatocyte-like colony resulting from coculture of BMCs and NPCs. (A) Fluorescence image, (B) bright-field image. The colony consisted of GFP-positive cells with round clear nuclei and granular cytoplasm, and the other GFP-positive cells with spindle or corpuscle-like morphology (C). Scale bars: 50 μm. Magnifications: A, ×100; B, ×100; C, ×200.
Journal of Hepatology  , 17-23DOI: ( /S (03) )

4. Fig. 3
Immunostaining of hepatocyte-like colonies. The cocultured BMCs and NPCs were stained with anti-CK8, CK18, and albumin antibodies. BMC-derived cells exhibiting GFP fluorescence (left). Hepatocyte-specific gene expression identified by red fluorescence (right). Merged images of hepatocyte-like colonies exhibiting both green and red fluorescence (middle). Scale bars: 50 μm. Original magnifications: A–C, G–I, ×100; D–F, ×200.
Journal of Hepatology  , 17-23DOI: ( /S (03) )

5. Fig. 4
Numbers of hepatocyte-like colonies in different culture conditions. BMCs and NPCs were cocultured in quadruplicate in the conditions listed in Table 1. Cells were fixed and immunostained with anti-CK8 antibody after 14 days. (A) The numbers of GFP- and CK8-positive colonies were counted by fluorescence microscopy. Results are means±SD of four dishes. (B) Numbers of hepatocyte-like colonies when cells were cultured in HSLF or in normal human sera from three healthy volunteers with AZA treatment, Dex, HGF, and OSM. Results are means±SD of four dishes in each condition. HSLF1, serum from the patient after living donor liver transplantation; HSLF2, serum from biliary atresia patient; NS, not significant.
Journal of Hepatology  , 17-23DOI: ( /S (03) )

6. Fig. 5
Detection of spontaneous cell fusion. BMCs from GFP transgenic mice and NPCs from the liver of LacZ transgenic mice were cocultured for 14 days. The cells were fixed and stained by X-gal and anti-CK8 immunocytochemistry. (A) BMC-derived cells exhibit GFP fluorescence. (B) Liver derived cells were stained as blue by X-gal substrate in bright-field image. Arrows indicate LacZ-positive cells. (C) CK8-expressing cells were identified by red fluorescence. CK8-positive cells had GFP fluorescence, whereas they were not stained by X-gal. Scale bars: 50 μm. Original magnifications: ×100.
Journal of Hepatology  , 17-23DOI: ( /S (03) )

7. Fig. 6
RT–PCR of BMC-derived cells. GFP-expressing BMC-derived cells were collected by FACS after 14 days of coculture. Total RNA was extracted and used for RT–PCR. (A) BMCs cultured under conditions for hepatocyte differentiation (condition A, left lane), expressed hepatocytes specific genes (albumin, TAT, CK8 and CK18) and cholangiocyte-specific gene (CK19), whereas those cells cultured in the absence of factors (condition B, middle lane) and freshly isolated BMCs (right lane) did not express any of these genes. Although AFP was detected in all cells, the BMCs cultured with NPCs (conditions A and B) had stronger expression than freshly isolated BMCs. (B,C) Quantitative RT–PCR of hepatocyte-specific genes (B, ALB; C, CK18) in BMC-derived cells cultured under different conditions. Quantification was performed by real time PCR using the Light Cycler system, and mRNA levels were normalized with respect to β-actin mRNA. Results are percentages of the mRNA formed in condition A.
Journal of Hepatology  , 17-23DOI: ( /S (03) )