Mature hepatocytes exhibit unexpected plasticity by direct dedifferentiation into liver progenitor cells in culture

Abstract

Although there have been numerous reports describing the isolation of liver progenitor cells from the adult liver, their exact origin has not been clearly defined; and the role played by mature hepatocytes as direct contributors to the hepatic progenitor cell pool has remained largely unknown. Here, we report strong evidence that mature hepatocytes in culture have the capacity to dedifferentiate into a population of adult liver progenitors without genetic or epigenetic manipulations. By using highly purified mature hepatocytes, which were obtained from untreated, healthy rat liver and labeled with fluorescent dye PKH2, we found that hepatocytes in culture gave rise to a population of PKH2-positive liver progenitor cells. These cells, liver-derived progenitor cells, which share phenotypic similarities with oval cells, were previously reported to be capable of forming mature hepatocytes, both in culture and in animals. Studies done at various time points during the course of dedifferentiation cultures revealed that hepatocytes rapidly transformed into liver progenitors within 1 week through a transient oval cell-like stage. This finding was supported by lineage-tracing studies involving double-transgenic AlbuminCreXRosa26 mice expressing β-galactosidase exclusively in hepatocytes. Cultures set up with hepatocytes obtained from these mice resulted in the generation of β-galactosidase-positive liver progenitor cells, demonstrating that they were a direct dedifferentiation product of mature hepatocytes. Additionally, these progenitors differentiated into hepatocytes in vivo when transplanted into rats that had undergone retrorsine pretreatment and partial hepatectomy.

Conclusion: Our studies provide strong evidence for the unexpected plasticity of mature hepatocytes to dedifferentiate into progenitor cells in culture, and this may potentially have a significant effect on the treatment of liver diseases requiring liver or hepatocyte transplantation.

Figure 1

Purity of rat hepatocytes isolated by low-G centrifugation. (A) RT-PCR analysis of the cell purity. The fresh liver cells were subjected to RT-PCR before (lane 1) and after (lane 2) low-G spin for the hepatocyte marker albumin, stellate cell marker desmin, endothelial cell marker vWF, Kupffer cell marker fucose receptor, and biliary ductal cell marker CK7. After low-G spin, only hepatocyte marker albumin was detectable. (B) Flow cytometric analysis of hepatocyte purity using an anti-albumin antibody. Over 99% of the cells were albumin-positive. (C) Immunofluorescence staining of the of the same cell population. Albumin is stained with green, HNF-1α with red and cell nuclei with blue (DAPI) fluorescence. In the merged image, virtually all cells were triple positive consistent with a highly pure hepatocyte preparation (original magnification: 100x).

Figure 2

Dedifferentiation of rat hepatocytes into LDPCs. (A) Morphology of LDPCs cultures initiated with PKH2-stained (cytoplasmic) hepatocytes at various time points during the culture period. The panels on the left are the bright-field images of the cultures on the indicated days, the panels on the right are the corresponding fluorescence images. PKH2 positive LDPCs began to emerge from hepatocytes starting on day 5. By day 14, virtually all the cells in the culture were LDPCs (original magnification: 100x). (B) Potential mechanisms by which hepatocytes dedifferentiate into LDPCs. The panel on the left shows hepatocytes shrinking or undergoing condensation (arrows) to become LDPCs. The panel on the right demonstrates a single multinucleated dedifferentiating hepatocyte giving rise to LDPCs (cell membranes are forming around the cell nuclei) by what appears to be fragmentation of the cytoplasm and budding off (arrows, original magnification: 200x). (C) Expression of mesenchymal markers CD44 and vimentin during the culture period. These markers which were not present in hepatocytes on Day 0 became detectable around Day 4 and on Day 12, they were strongly positive in LDPCs suggesting that hepatocytes underwent an EMT during their transformation to LDPCs (original magnification: 100x).

Figure 3

Analysis of hepatocyte-specific and LDPC-specific markers at various time points during the LDPC culture period. (A) RT-PCR for the hepatocyte markers; albumin and HNF-1α, and LDPCs markers; LMO2, CD45. On day 0, only hepatocyte markers were expressed and no signals for LDPC markers were detectable. Beginning around day 4, hepatocyte markers became weaker and virtually gone by day 8 while LDPC markers showed an opposite trend and became progressively stronger. Day 12 cultures and “pure” LDPCs obtained on day 14 by gentle EDTA treatment of the cultures showed no difference indicating that the transformation into LDPCs was completed (only the cell number increased after day 12). The lane marked Neg. shows the negative control reaction. (B) IF studies of the LDPC cultures for the same markers confirmed the RT-PCR results again showing rapid disappearance of hepatocyte markers by day 4 and progressively stronger expression of LDPC markers after day 8 (cell nuclei stained by DAPI in blue, original magnification, 100x). The observed patterns were consistent with the rapid transformation of hepatocytes into LDPCs observed morphologically in Figure 2.

Figure 4

Transition of dedifferentiating hepatocytes through an oval cell-like stage en route to LDPCs. (A) RT-PCR analysis of two oval cell markers (also expressed by biliary ductal cells) CK7 and GGT at various time points during LDPC culture period. Both markers, which were not expressed initially, became detectable between days 4 and 6 at about the same time as the loss of hepatocyte-specific markers shown in Figure 3. They then peaked and subsequently became undetectable in “pure” LDPCs collected on day 14. The lane marked Neg. shows the negative control reaction. (B) IF analysis of the LDPC cultures during the same period for CK7, GGT and oval cell-specific protein OV-6. The expression pattern for CK7 and GGT confirms the pattern seen with RT-PCR analysis with the exception that GGT expression persisted in LDPCs at low level (most likely previously transcribed protein). OV-6 expression, on the other hand became detectable on day 6, reached its peak on day 8 and then gradually declined to undetectable levels in LDPCs on day 12 (original magnification, 100x). (C) Co-staining of the cells for LDPC markers CD45 and LMO2 and oval cell marker OV-6 on day 8 of the culture. Virtually all cells co-expressed LDPC markers and oval cell marker indicating a transient overlap between oval cell-like stage and LDPC stage. The combined data from RT-PCR and IF staining strongly suggests that hepatocytes transition through an oval cell-like stage before they turn into LDPCs (original magnification: 100x).

Figure 5

β-galactosidase expression in double transgenic AlbCreXRosa26 mice. (A) Western blot analysis of β-galactosidase expression. The blot showed that double transgenic kidney (lane 1), double transgenic spleen (lane 4) and wild-type liver (lane 3) did not express β-galactosidase where as double transgenic liver (lane 2) was positive. (B) X-gal and β-galactosidase IF staining of the double transgenic liver. X-gal staining of the wild-type liver did not show any reaction (left upper panel, original magnification: 100x) where as double transgenic liver strongly reacted. X-gal positivity appeared to be restricted to hepatocytes of the double transgenic mice as some cells including vascular endothelium (arrow) were x-gal negative (right upper panel, original magnification: 100x). Immunofluorescent co-staining for β-galactosidase and albumin (left lower panel, original magnification: 100x) showed complete overlap of the markers. On the other hand CK7 positive ductal cells (arrow) did not express β-galactosidase (right lower panel, original magnification: 200x). Overall data were consistent with tissue-specific (liver) and cell-specific (hepatocyte) expression of β-galactosidase in AlbCreXRosa26 mice.

Figure 6

Analysis of β-galactosidase expression in LDPCs derived from double transgenic AlbCreXRosa26 mice. (A) X-gal staining of LDPC cultures on day 0 showed that nearly all hepatocytes were x-gal positive. On Day 4, dedifferentiating hepatocytes began to give rise to small x-gal positive cells which become more prominent by Day 8. On Day 15, virtually all cells in the culture were x-gal positive LDPCs (original magnification in all panels: 100x). (B) Confirmation of β-galactosidase positive cells as LDPCs. The round small cells that emerged in the cultures set up with hepatocytes from AlbCreXRosa26 mice were subjected to co-staining for β-galactosidase and LDPC markers CD45 and LMO2 (original magnification in all panels: 100x). The results showed complete overlap of the β-galactosidase and LDPC markers confirming that the cells were indeed LDPCs expressing β-galactosidase. These findings strongly support the hypothesis that LDPCs directly originate from hepatocytes.

Figure 7

Hepatic differentiation of LDPCs in vivo. Fluorescent-labeled (PKH26) LDPCs from male Fischer rats were transplanted into female Fischer rats that had undergone retrorsine pretreatment and partial hepatectomy. Frozen liver section obtained from the recipient rats two months after the transplantation were examined under fluorescence microscope. (a) Unstained section showing PKH + cells, (b) DAPI nuclear staining, (c) merged images of a and b (original magnification: 100x). They showed foci of PKH+ cells morphologically consistent with biliary ductal cells and hepatocytes. To confirm that male LDPCs had engrafted and differentiated into hepatocytes in the recipients, we performed IF staining for albumin and FISH for Y-chromosome. (d) Merged images of DAPI nuclear staining and FISH (green dot) for Y-chromosome, (e) merged images of albumin staining (red) and FISH for Y-chromosome, (f) merged images of d and e (original magnification: 400x). These findings showed that transplanted LDPCs had engrafted in the liver and differentiated into hepatocytes