Time course of expression and nuclear localization of HNF4α. Lin⁻OSMRβ⁺ cells were cultured in the presence of transdifferentiation medium. The expression and nuclear localization of HNF4α were analyzed at different times. A, immunocytochemical analysis of HNF4α in OSM-neutralized culture. Cells were cultured without or with OSM-neutralizing antibody. Control cells show the expression of HNF4α in the majority of the cells (nuclei stained with 4′,6-diamidino-2-phenylindole are pseudocolored in red; left panel). HNF4α expression and nuclear localization were reduced in the presence of the OSM-neutralizing antibody (right panel) (n = 3). B, flow cytometric analysis of albumin expression. The bar diagram shows the amount of albumin-expressing cells in the respective cultures. The cellular differentiation process was suppressed (green bar) in OSM-neutralized medium compared with the DLS control culture (black bar). The red bar (background reading) indicates cells that were cultured in the normal serum (NS)-supplemented medium (n = 3). C, Western blot analysis of HNF4α expression. Cytoplasmic extract (CE) and nuclear extract (NE) of cells at different time intervals were probed with anti-HNF4α, anti-actin, and anti-histone H3 antibodies. A strong signal for nuclear localization of HNF4α was observed after 7 days of culture. Fold expression/activation of HNF4α are shown as normalized values with respect to cytoplasmic and nuclear proteins (supplemental Table 2). HNF4α, actin, and histone H3 migrate as 55-, 43-, and 17-kDa proteins, respectively. D, immunocytochemical analysis of nuclear localization of HNF4α. Representative confocal photomicrographs show that HNF4α was expressed from day 3 and increased on day 5 of culture. High expression of albumin was observed on day 7 of culture. Arrows indicate nuclear localization of HNF4α.

HNF4α-transfected 32D cells synthesize hepatocyte-specific proteins. HNF4α and hepatic proteins were analyzed by immunocytochemistry using specific antibodies. Representative micrographs show the presence of HNF4α in the nuclei of cells (b, f, and j) that express albumin (c), α-fetoprotein (g), and CK-18 (k). The induced, differentiated cells expressed the P450 enzyme because the fluorescent compound resorufin (n) was produced from 7-pentoxyresorufin (n = 3).

Kinetics of differentiation of BM cells into hepatic cells. Lin⁻OSMRβ⁺ cells (one cell/well) were cultured in 96-well plates for different times in the presence of transdifferentiation medium (see supplemental “Methods”). Half of the medium was replaced twice in a week. Twenty-five wells for each time point were examined. A, quantitative analysis (immunocytochemistry-based) of cells expressing CD45, CD45 and albumin, or albumin. Red bars, CD45⁺Alb⁻ cells; yellow bars, CD45⁺Alb⁺ cells; green bars, CD45⁻Alb⁺ cells. Results show that hematopoietic cells were transdifferentiated into albumin-expressing hepatic cells after 7 days of culture. Note that the results of all of the wells were not reported because a few wells in each time point showed either no cells or no staining with the CD45/Alb antibodies. B, confocal microscopic analysis of OSMRβ, c-Met, and albumin. Representative photomicrographs show that OSMRβ was initially expressed in the cells but not c-Met. After 5 days of culture, cells expressed OSMRβ, c-Met, and albumin. After day 5, the expression of OSMRβ was completely down-regulated. The number of experiments (n) was 2.

Ectopic expression of HNF4α induces differentiation of 32D cells. The transfected cells were analyzed for the expression of cell surface receptors, colony-forming ability, and gene expression. A, functional activation of c-Met in FL-HNF4α-transfected cells. In the transfected cells that expressed c-Met (b), the number of BrdUrd-incorporated cells was lower in the absence of HGF (e) than in its presence (h) (n = 3). B, RT-PCR analysis of gene expression in FL-HNF4α-transfected cells. The HNF1α, HNF4α, and albumin genes were expressed in the transfected cells, whereas GATA1 was completely down-regulated. AML-12 (mouse primary hepatocytes) were used as a positive control for hepatic genes (n = 2). HNF4α, HNF1α, HNF3β, c/EBPα, GATA1, albumin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) migrate as 180-, 187-, 216-, 314-, 187-, 465-, and 209-bp DNA fragments, respectively. C, myeloid colony assay (42). HNF4α transfection caused a significant drop in the colony-forming potential of the cells (n = 3).

Ectopic expression of DN-HNF4α in Lin⁻OSMRβ⁺ cells inhibits transdifferentiation. Lin⁻OSMRβ⁺ cells were cultured and transfected with the EV or DN-HNF4α plasmids for 7 days. Comparative mRNA expression of albumin (left) and tyrosine aminotransferase (TAT; right) are shown in representative agarose gel pictures. First lane, purified Lin⁻OSMRβ⁺ cells (negative control); second lane 2, Lin⁻OSMRβ⁺ cells transfected with EV (vector control); third lane, Lin⁻OSMRβ⁺ cells transfected with the DN-HNF4α plasmid (test sample); fourth lane, AML-12 cells (positive control). Albumin, tyrosine aminotransferase (TAT), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) migrate as 465-, 422-, and 209-bp DNA fragments, respectively. Intensities of respective bands were quantified with the ImageJ software. The background intensity in the first lane was subtracted from that of the other three lanes. The absolute readings were normalized with respect to the GAPDH band. Bar diagrams show the relative transcription levels of the albumin and tyrosine aminotransferase genes (n = 2).