Hidekazu Ooe

Proliferation of hepatocyte progenitor cells isolated from adult human livers in serum-free medium.

Rat small hepatocytes (SHs) are committed progenitor cells that can differentiate into mature hepatocytes and can selectively proliferate in serum-free medium when they are cultured on hyaluronic acid (HA)-coated dishes. In this study we examined the separation of human SHs from adult human livers. We obtained liver tissues from the resected liver of 16 patients who underwent hepatic resections. Extracted liver specimens were clearly separate from the tumor regions with sufficient margins. Hepatic cells were isolated using the modified method of two-step collagenase perfusion. A low-speed centrifugation was performed and cells in the supernatant were finally cultured on HA-coated dishes in serum-free DMEM/F12 medium including nicotinamide, EGF, and HGF. Small-sized hepatocytes selectively proliferated to form colonies and many colonies continued growing for more than 3 weeks. The average number of cells in a colony was 38.6 ± 18.0, 79.0 ± 54.0, and 101.5 ± 115.7 at day 7, 14, and 21, respectively. About 0.04% of plated cells could form an SH colony. Immunocytochemistry showed that the cells forming a colony were positive for albumin, transferrin, keratin 8, and CD44. The results of RT-PCR showed that colony-forming cells expressed albumin, transferrin, α1-antitrypsin, fibrinogen, glutamine synthetase, many cytochrome P450s, and liver-enriched transcription factors (HNF3α, HNF4α, C/EBPα, and C/EBPβ). Furthermore, the cells expressed not only the genes of hepatic differentiated functions but also those of both hepatic stem cell marker (Thy1.1, EpCAM, AFP) and SH marker (CD44, D6.1A, BRI3). Albumin secretion into culture medium was also observed. Our results demonstrate the existence of hepatocyte progenitor cells in human adult livers, and the cells can grow in a serum-free medium on HA-coated dishes. Human SHs may be a useful source for cell transplantation as well as pharmaceutical and toxicological investigations.

Selective proliferation of rat hepatocyte progenitor cells in serum-free culture

This protocol details a method of obtaining selectively proliferated hepatocyte progenitor cells using hyaluronic acid (HA)–coated dishes and serum-free medium. A small hepatocyte (SH) is a hepatocyte progenitor cell of adult livers and has many hepatic functions. When the rat SH begins to proliferate, CD44 is specifically expressed. To define the purification of SH, CD44 and cytokeratin 8 are used as marker proteins. The growth of SHs is faster on HA-coated dishes than on other extracellular matrix–coated ones. The use of both DMEM/F12 medium and HA-coated dishes allows the selective proliferation of SHs in culture. The purification of SHs is approximately 85% at day 10.

Thy1-Positive Cells Have Bipotential Ability to Differentiate into Hepatocytes and Biliary Epithelial Cells in Galactosamine-Induced Rat Liver Regeneration

In galactosamine (GalN)-induced rat liver injury, hepatic stem/progenitor cells, small hepatocytes (SHs) and oval cells, transiently appear in the initial period of liver regeneration. To clarify the relationship between SHs and oval cells, CD44(+) and Thy1(+) cells were sorted from GalN-treated livers and used as candidates for SHs and oval cells, respectively. Some Thy1(+) cells isolated 3 days after GalN-treatment (GalN-D3) formed CD44(+) cell colonies, but those from GalN-D2 could form few. GeneChip (Affymetrix, Inc, Santa Clara, CA) analysis of the sorted cells and cultured Thy1(+) cells suggested that hepatocytic differentiation progressed in the order Thy1(+) (GalN-D3), Thy1(+) cell colony (Thy1-C), and CD44(+) (GalN-D4) cells. When Thy1(+), Thy1-C, and CD44(+) cells were transplanted into retrorsine/PH rat livers, they could proliferate to form hepatocytic foci. At 30 days after transplantation most cells forming the foci derived from CD44(+) cells possessed C/EBPalpha(+) nuclei, whereas only a few cells derived from Thy1-C showed this positivity. When Thy1(+) (GalN-D3) cells were cultured between collagen gels in medium with hepatocyte growth factor(+)/dexamethasone(-)/dimethyl sulfoxide(-), ducts/cysts consisting of biliary epithelial cells appeared, whereas with CD44(+) and Thy1(+) (GalN-D2) cells they did not. Taken together, these results indicate that the commitment of Thy1(+) cells to differentiate into hepatocytes or biliary epithelial cells may occur between Day 2 and Day 3. Furthermore, some Thy1(+) cells may differentiate into hepatocytes via CD44(+) SHs.

Functional Expression of Organic Anion Transporters in Hepatic Organoids Reconstructed by Rat Small Hepatocytes

Small hepatocytes (SHs) are hepatic progenitor cells with hepatic characteristics. They can proliferate to form colonies in culture and change their morphology from flat to rising/piled‐up with bile canaliculi (BC), which results in maturation. In this study, we examined whether SHs could express hepatic transporters with polarity, whether the transporters could transport organic anion substrates into BC, and whether the secreted substances could be recovered from BC. Immunocytochemistry and RT‐PCR were carried out. [3H]‐labeled estrogen derivatives were used to measure the functions of the transporters in SHs isolated from normal and multidrug resistance‐associated protein (Mrp) 2‐deficient rats. The results showed that organic anion‐transporting proteins (Oatps) 1 and 2, Na+‐dependent taurocholate co‐transporting polypeptide (Ntcp), Mrp2, and bile‐salt export pump (Bsep) were well expressed in rising/piled‐up cells and that their expression was correlated to that of hepatocyte nuclear factor 4α. Although small SHs expressed not Oatps and Mrp2 but Mrp3, rising/piled‐up SHs expressed Oatp1 and 2 and Mrp2 proteins in the sinusoidal and BC membranes, respectively. On the other hand, breast cancer resistant protein (Bcrp) and Mrp3 expression decreased as SHs matured. The substrate transported via Oatps and Mrp2 was secreted into BC and it accumulated in both BC and cyst‐like structures. The secreted substrate could be efficiently recovered from BC reconstructed by SHs derived from a normal rat, but not from an Mrp2‐deficient rat. In conclusion, SHs can reconstitute hepatic organoids expressing functional organic anion transporters in culture. This culture system may be useful to analyze the metabolism and excretion mechanisms of drugs. J. Cell. Biochem. 104: 68–81, 2008.

Differentiation capacity of hepatic stem/progenitor cells isolated from D-galactosamine-treated rat livers.

Oval cells and small hepatocytes (SHs) are known to be hepatic stem and progenitor cells. Although oval cells are believed to differentiate into mature hepatocytes (MHs) through SHs, the details of their differentiation process are not well understood. Furthermore, it is not certain whether the induced cells possess fully mature functions as MHs. In the present experiment, we used Thy1 and CD44 to isolate oval and progenitor cells, respectively, from D‐galactosamine‐treated rat livers. Epidermal growth factor, basic fibroblast growth factor, or hepatocyte growth factor could trigger the hepatocytic differentiation of sorted Thy1+ cells to form epithelial cell colonies, and the combination of the factors stimulated the emergence and expansion of the colonies. Cells in the Thy1+‐derived colonies grew more slowly than those in the CD44+‐derived ones in vitro and in vivo and the degree of their hepatocytic differentiation increased with CD44 expression. Although the induced hepatocytes derived from Thy1+ and CD44+ cells showed similar morphology to MHs and formed organoids from the colonies similar to those from SHs, many hepatic differentiated functions of the induced hepatocytes were less well performed than those of mature SHs derived from the healthy liver. The gene expression of cytochrome P450 1A2, tryptophan 2,3‐dioxygenase, and carbamoylphosphate synthetase I was lower in the induced hepatocytes than in mature SHs. In addition, the protein expression of CCAAT/enhancer‐binding protein alpha and bile canalicular formation could not reach the levels of production of mature SHs. Conclusion: The results suggest that, although Thy1+ and CD44+ cells are able to differentiate into hepatocytes, the degree of maturation of the induced hepatocytes may not be equal to that of healthy resident hepatocytes. (HEPATOLOGY 2013)

Cytochrome P450 Expression of Cultured Rat Small Hepatocytes After Long-Term Cryopreservation

Small hepatocytes (SHs) are hepatic progenitor cells that can be cryopreserved for a long time. After thawing, the cells can proliferate and, when treated with Matrigel, they can differentiate into mature hepatocytes (MHs). In this study, we investigated whether cryopreserved SHs could express cytochromes P450 (P450s), whether P450 expression was induced by appropriate inducers, and whether P450 activities were measurable. 3-Methylcholanthrene (3-MC), phenobarbital (PB), pregnenolone-16α-carbonitrile (PCN), and ethanol were used as inducers for CYP1A, 2B, 3A, and 2E, respectively. Immunoblot analysis indicated that cryopreserved SHs constitutively expressed CYP1A1/2, CYP2E1, and CYP3A2 as much as 26 days after plating. Significant expression of CYP1A1/2 and 3A2 in the cells treated with Matrigel was induced by 3-MC and PCN, respectively. Although Matrigel did not up-regulate the enzymatic activity of CYP1A, CYP3A and CYP2E activities increased. Induction of CYP1A and CYP3A activities by each inducer was observed in cryopreserved cells treated with Matrigel. Although the expression of CYP2B1 could be detected in subcultured SHs treated with PB, it was not detected in cryopreserved SHs. The activity of NADPH-cytochrome P450 reductase was measured in both subcultured and cryopreserved SHs, although the activities in both were approximately 30% of that of MHs. Profiles of 14C-testosterone metabolites were examined in cultured MHs and in cryopreserved SHs by high-performance liquid chromatography. Similar peaks for testosterone metabolites in MHs and SHs were observed in the same elution time. These results indicate that, although induction of CYP3A and 2B in cryopreserved SHs is inferior to that in subcultured ones, SHs can maintain the expression and activities of P450s after long-term cryopreservation.

Growth Ability and Repopulation Efficiency of Transplanted Hepatic Stem Cells, Progenitor Cells, and Mature Hepatocytes in Retrorsine-Treated Rat Livers:

Cell-based therapies as an alternative to liver transplantation have been anticipated for the treatment of potentially fatal liver diseases. Not only mature hepatocytes (MHs) but also hepatic stem/progenitor cells are considered as candidate cell sources. However, whether the stem/progenitor cells have an advantage to engraft and repopulate the recipient liver compared with MHs has not been comprehensively assessed. Therefore, we used Thy1+ (oval) and CD44+ (small hepatocytes) cells isolated from GalN-treated rat livers as hepatic stem and progenitor cells, respectively. Cells from dipeptidylpeptidase IV (DPPIV)+ rat livers were transplanted into DPPIV” livers treated with retrorsine following partial hepatectomy. Both stem and progenitor cells could differentiate into hepatocytes in host livers. In addition, the growth of the progenitor cells was faster than that of MHs until days 14. However, their repopulation efficiency in the long term was very low, since the survival period of the progenitor cells was much shorter than that of MHs. Most foci derived from Thy1+ cells disappeared within 2 months. Many cells expressed senescence-associated β-galactosidase in 33% of CD44-derived foci at day 60, whereas the expression was observed in 13% of MH-derived ones. The short life of the cells may be due to their cellular senescence. On the other hand, the incorporation of sinusoidal endothelial cells into foci and sinusoid formation, which might be correlated to hepatic maturation, was completed faster in MH-derived foci than in CD44-derived ones. The survival of donor cells may have a close relation to not only early integration into hepatic plates but also the differentiated state of the cells at the time of transplantation.

Thyroid Hormone Is Necessary for Expression of Constitutive Androstane Receptor in Rat Hepatocytes

Small hepatocytes are hepatocyte progenitor cells that possess the capability of maturation and cryopreservation. When cryopreserved rat small hepatocytes were cultured in serum-free medium, the protein expression and the inducibility of CYP1A1/2, CYP2E1, and CYP3A were maintained, but those of CYP2B1 were lost. In this study we investigated the cause of the loss of CYP2B1 expression in cryopreserved small hepatocytes by reverse transcription-polymerase chain reaction, immunoblotting, and chromatin immunoprecipitation assay. Expression of mRNA and protein of the nuclear receptor, constitutive androstane receptor (CAR), which regulates the expression of CYP2B1, was inhibited in the serum-free culture of cryopreserved small hepatocytes, whereas they were expressed in that of subcultured small hepatocytes. Serum application dramatically induced CAR expression in the culture of cryopreserved small hepatocytes. The addition of very low concentrations of thyroid hormones (THs; 3,5,3′-triiodothyronine, 5 × 10–12 M; thyroxine, 5 × 10–12-5 × 10–10 M) to the medium also induced the expression of CAR and CYP2B1. Moreover, CYP2B1 expression was induced by administration of phenobarbital. In rats with hypothyroidism induced by thyroidectomy and 6-propyl-2-thiouracil treatment, the expression of CAR and CYP2B1 was strongly repressed. Although THs do not directly regulate the expression of CAR, they may be important for rat hepatocytes to regulate CYP2B1 through CAR expression in the physiological condition.

Proliferation of rat small hepatocytes requires follistatin expression.

Small hepatocytes (SHs) are a subpopulation of hepatocytes that have high growth potential in culture and can differentiate into mature hepatocytes (MHs). The activin (Act)/follistatin (Fst) system critically contributes to homeostasis of cell growth in the normal liver. ActA and ActB consist of two disulfide‐linked Inhibin (Inh)β subunits, InhβA and InhβB, respectively. Fst binds to Act and blocks its bioactivity. In the present study we carried out the experiments to clarify how Fst regulates the proliferation of SHs. The gene expression was analyzed using DNA microarray analysis, reverse transcription‐polymerase chain reaction (RT‐PCR) and real‐time PCR, and protein expression was examined by western blots, immunocytochemistry, and enzyme‐linked immunosorbent assay. RT‐PCR showed that Fst expression was high in SHs and low in MHs. Although the ActA expression was opposite to that of Fst, ActB expression was high in SHs and low in MHs and increased with time in culture. Fst protein was detected in the cytoplasm of SHs and secreted into the culture medium. ActB protein was also secreted into the medium. Although the exogenous administration of ActA and ActB apparently suppressed the proliferation of SHs, apoptosis of SHs was not induced by treatment with ActA or ActB. On the other hand, Fst treatment did not affect the colony formation of SHs but prevented the inhibitory effect of ActA. Neutralization by the anti‐Fst antibody resulted in the suppression of DNA synthesis in SHs, and small hairpin RNA against Fst suppressed the expansion of SH colonies. In conclusion, Fst expression is necessary for the proliferation of SHs. J. Cell. Physiol. 227: 2363–2370, 2012.

Characterization of hepatic-organoid cultures.

Small hepatocytes (SHs) are “committed progenitor cells” that can further differentiate into mature hepatocytes (MHs). SHs can proliferate to form colonies, and the maturation of SHs occurs with the alteration of the cell shape from small and flat to large and rising/piled-up. The hepatic organoids consisting of rising/piled-up cells possess highly differentiated functions like those of MHs and anastomosing networks of bile canaliculi (BC) are developed. The cells can make bile, secrete it into BC, and the bile can be carried without leaking. Thus, the organoids consist of polarized hepatocytes and possess biochemical and physiological functions as hepatic tissue.