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

我们发现三个基因CD44、BRI3和D6.1A在小肝细胞（SHs）中的表达比在成熟肝细胞（MHs）中的表达高得多，并且具有跨膜结构域。利用CD44、BRI3和D6.1A特异性抗体，我们检测了SHs在大鼠肝脏中的定位。虽然在正常肝脏的肝小叶中未观察到这些蛋白的表达，但在半乳糖胺处理的严重损伤肝门静脉周围病变中存在与抗体反应的肝细胞。利用带磁珠抗体（MACS），从半乳糖胺处理的肝脏中分选CD44+细胞，然后进行培养。CD44+细胞可以增殖并形成集落，其形态与SHs相似。虽然肝脏标记基因在分选和培养的CD44+细胞中均有表达，但在胆管细胞和卵圆细胞中均未检测到标记基因。将培养的SHs结合放射和部分门静脉结扎术移植到遗传大鼠肝脏中，SHs插入肝小梁并增殖。为了重建肝类器官，我们尝试了三种不同的方法。(1)将SHs和NPCs分别涂于胶原海绵上后，SHs可在海绵中增殖扩张形成肝类器官。该结构包括具有胆管、胆管和毛细血管样结构的mhh。此外，我们还观察到分离的人肝细胞可以在海绵中形成肝类器官。(2) SHs在两层多孔膜上培养至形成大菌落后，一层膜堆叠在另一层膜上，使细胞面对面。堆积的细胞可分化为MHs，形成胆小管网络。(3)将肝细胞分离后的SH菌落和残体结缔组织在微重力旋转培养系统中培养，几周即可形成较大尺寸的肝类器官。

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.