Identification of specific surface proteins in small hepatocytes and reconstruction of hepatic organoids ex vivo.

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We found three genes, CD44, BRI3, and D6.1A, with much higher expression in small hepatocytes (SHs)than in mature hepatocytes (MHs)and possessing transmembrane domain. Using specific antibody to CD44, BRI3, and D6.1A, we examined the localization of SHs in a rat liver. Although none of expressions of the proteins were observed in hepatic lobules of a normal liver, the hepatocytes reacting with the antibodies existed in the periportal lesion of severely injured liver treated with galactosamine. Using the antibody with magnetic beads (MACS), CD44+ cells were sorted from the galactosamine-treated livers and then cultured. The CD44+ cells could proliferate and form colonies, which are morphologically similar to SHs. Although hepatic marker genes were expressed in both sorted and cultured CD44+ cells, neither bile duct cell nor oval cell marker genes were detected by PCR. When cultured SHs were transplanted into congenic rat livers in combination with radiation and partial portal ligation, SHs were inserted into hepatic trabecules and proliferated.To reconstruct hepatic organoids, we tried three different methods. (1)When SHs and NPCs were plated on collagen sponge, SHs could proliferate and expand to form a hepatic organoid in the sponge. The structure includes MHs with bile canaliculi, bile ducts, and capillary-like structures. Furthermore, we also observed that isolated human hepatic cells could form hepatic organoid in the sponge. (2)After SHs were cultured on two multiporous membranes until they formed large colonies, one membrane were stacked on the other membrane as cells faced each other. The stacked cells could differentiate into MHs with forming bile canalicular networks. (3)When both SH colonies and the connective tissues of the remnant after the hepatocyte isolation were cultured in the microgravity rotating culture system, a relatively large size of hepatic organoids was formed with a few weeks.