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Differentiated epithelial cells show substantial lineage plasticity upon severe tissue injuries. In chronically injured mouse livers, part of hepatocytes become Sry-HMG box containing 9 (Sox9) (+) epithelial cell adhesion molecule (-) hepatocyte nuclear factor 4 α (+) biphenotypic hepatocytes. However, it is not clear whether all Sox9 + hepatocytes uniformly possess cellular properties as hepatocyte progenitors. Here, we examined the microarray data comparing Sox9 + hepatocytes with mature hepatocytes and identified CD24 as a novel marker for biphenotypic hepatocytes. Immunohistochemical analyses showed that part of Sox9 + hepatocytes near expanded ductular structures expressed CD24 in the liver injured by 3,5-diethoxycarbonyl-1,4-dihydro-collidine (DDC) diet and by bile duct ligation. Indeed, Sox9 + hepatocytes could be separated into CD24 - and CD24 + cells by fluorescence activated cell sorting. The ratio of CD24 + cells against CD24 - ones in Sox9 + hepatocytes gradually increased while DDC-injury progressed and colony-forming capability mostly attributed to CD24 + cells. Although hepatocyte markers were remarkably downregulated in of Sox9 + CD24 + hepatocytes, they re-differentiated into mature hepatocytes in vitro and in vivo. Our current results demonstrate that the emergence of biphenotypic hepatocytes is a sequential event including the transition from CD24 - and CD24 + status, which may be a crucial step for hepatocytes to acquire progenitor properties.

It has been proposed that tissue stem cells supply multiple epithelial cells in mature tissues and organs. However, it is unclear whether tissue stem cells generally contribute to cellular turnover in normal healthy organs. Here, we show that liver progenitors distinct from bipotent liver stem/progenitor cells (LPCs) persistently exist in mouse livers and potentially contribute to tissue maintenance. We found that, in addition to LPCs isolated as EpCAM + cells, liver progenitors were enriched in CD45 - TER119 - CD31 - EpCAM - ICAM-1 + fraction isolated from late-fetal and postnatal livers. ICAM-1 + liver progenitors were abundant by 4 weeks (4W) after birth. Although their number decreased with age, ICAM-1 + liver progenitors existed in livers beyond that stage. We established liver progenitor clones derived from ICAM-1 + cells between 1 and 20W and found that those clones efficiently differentiated into mature hepatocytes (MHs), which secreted albumin, eliminated ammonium ion, stored glycogen, and showed cytochrome P450 activity. Even after long-term culture, those clones kept potential to differentiate to MHs. When ICAM-1 + clones were transplanted into nude mice after retrorsine treatment and 70% partial hepatectomy, donor cells were incorporated into liver plates and expressed hepatocyte nuclear factor 4α, CCAAT/enhancer binding protein α, and carbamoylphosphate synthetase I. Moreover, after short-term treatment with oncostatin M, ICAM-1 + clones could efficiently repopulate the recipient liver tissues. Our results indicate that liver progenitors that can efficiently differentiate to MHs exist in normal adult livers. Those liver progenitors could be an important source of new MHs for tissue maintenance and repair in vivo, and for regenerative medicine ex vivo. Stem Cells 2016;34:2889-2901., (© 2016 AlphaMed Press.)

Late fetal and adult livers are reported to contain bipotential liver stem/progenitor cells (LPCs), which share surface markers, including epithelial cell adhesion molecule (EpCAM), with cholangiocytes and differentiate into both hepatocytes and cholangiocytes. However, recent results do not necessarily support the idea that LPCs contribute significantly to cellular turnover and regeneration by supplying new hepatocytes. Here, we examined the colony-forming capability of EpCAM(+) cells isolated from mouse livers between E17 and 11 weeks of age. We found that the number of bipotential colonies was greatly reduced between 1 and 6 weeks, indicating that the number of LPCs decreases during postnatal development. Moreover, bipotential colonies derived from adult LPCs contained substantially fewer albumin(+) cells than those from neonatal LPCs. We further examined the differentiation potential of neonatal and adult LPCs by transplantation and found that neonatal cells differentiated into mature hepatocytes in recipient livers more frequently than adult LPCs. Since we previously reported that the transcription factor grainyhead-like 2 (GRHL2) expressed in EpCAM(+) cells inhibits hepatocytic differentiation, we examined whether targets of GRHL2 might block hepatocytic differentiation. DNA and microRNA microarrays revealed that miR122, the expression of which correlates with hepatocytic differentiation, was greatly reduced in adult as compared with neonatal EpCAM(+) cells. Indeed, GRHL2 negatively regulates the promoter/enhancer activity of the Mir122 gene. Our results indicate that neonatal but not adult EpCAM(+) LPCs have great potential to produce albumin(+) hepatocytes. GRHL2 suppresses transcription of miR122 and thereby restricts the differentiation potential of adult LPCs., (© 2014. Published by The Company of Biologists Ltd.)

In developing organs, epithelial tissue structures are mostly developed by the perinatal period. However, it is unknown whether epithelial cells are already functionally mature and whether they are fixed in their lineage. Here we show that epithelial cells alter their plasticity during postnatal development by examining the differentiation potential of epithelial cell adhesion molecule (EpCAM)(+) cholangiocytes (biliary epithelial cells) isolated from neonatal and adult mouse livers. We found that neonatal cholangiocytes isolated from 1-week-old liver converted into functional hepatocytes in the presence of oncostatin M and Matrigel®. In contrast, neither morphological changes nor expression of hepatocyte markers were induced in adult cholangiocytes. The transcription factors hepatocyte nuclear factor 4α and CCAAT/enhancer binding protein α (C/EBPα), which are necessary for hepatocytic differentiation, were induced in neonatal cholangiocytes but not in adult cells, whereas grainyhead-like 2 (Grhl2) and hairy-enhance of slit 1 (Hes1), which are implicated in cholangiocyte differentiation, were continuously expressed in adult cells. Overexpression of C/EBPα and Grhl2 promoted and inhibited hepatocytic differentiation, respectively. Furthermore, adult cholangiocytes formed a monolayer with higher barrier function than neonatal ones did, suggesting that cholangiocytes are still in the process of epithelial maturation even after forming tubular structures during the neonatal period. Taken together, these results suggest that cholangiocytes lose plasticity to convert into hepatocytes during epithelial maturation. They lose competency to upregulate hepatocytic transcription factors and downregulate cholagiocytic ones under conditions inducing hepatocytic differentiation. Our results suggest that a molecular machinery augmenting epithelial integrity limits lineage plasticity of epithelial cells.

The objective of this study is to investigate the attachment, osteogenic differentiation, and collagen matrix secretion of rat bone marrow stromal cells (MSC) and MC3T3-E1 cells on the biological apatite (Ap) with different crystalline facets. The Ap samples with a-facet and c-facet surfaces were prepared by cutting the heat-treated cortical bone of bovine femurs parallel and perpendicular to the longitudinal direction, respectively. MC3T3-E1 cells attached spreading on the a-Ap sample whereas their shape of cells was round on the c-Ap. No difference in the proliferation profile of MSC and MC3T3-E1 cells was observed between the two samples. The activity of alkaline phosphatase was significantly higher for both the cells on the a-Ap sample than that of c-Ap one. The matrix of collagen secreted from MC3T3-E1 cells on the a-Ap sample oriented along the c-axis of Ap, in contrast to those of c-Ap. The cells tended to align along the collagen orientation. It is concluded that the alignment of cells is influenced by the collagen fibril orientation of Ap samples., (Copyright 2009 Wiley Periodicals, Inc.)

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.

The proliferation, osteogenic differentiation, and distribution patterns of stromal cells from rat bone marrow were investigated in a three-dimensional nonwoven fabric of polyethylene terephthalate fiber by the static, agitated, and stirred culture methods; stirring speeds were 10, 50, and 100 rpm in the stirred culture method. The culture method affected the time profile of proliferation and osteogenic differentiation of cells or their distribution in the fabric. The extent of cell proliferation and osteogenic differentiation became higher in order of the stirred at 100 rpm = the stirred at 50 rpm > the stirred at 10 rpm > the agitated > the static methods. In addition, the cells were more uniformly proliferated in the fabric by the stirred culture method with time than they were proliferated in the fabric by other methods. The alkaline phosphatase (ALP) activity and calcium content were higher for cells cultured by the stirred culture method than those cultured by other methods. The total ALP activity, calcium content, and bone mineral density were higher for every stirred method than those for other methods. However, the distribution uniformity of cells differentiated was low irrespective of the culture method. It is concluded that the extent of proliferation and differentiation of cells or their distribution uniformity in the nonwoven fabrics was influenced by the culture method.