Research for the formation of hepatic organoids by using human hepatic progenitor cells and the model of artificial liver device incorporated with human hepatic organoids

我们报道了CD44在大鼠小肝细胞中特异性表达，CD44^+肝细胞在损伤大鼠肝脏中短暂出现。利用一种特异性抗体，我们从半乳糖胺处理过的大鼠肝脏中分离出CD44^+细胞并进行培养。细胞的表型和基因表达与培养的SHs相似，^+细胞可以分化为肝细胞（J Hepatol, 2006）。此外，半乳糖胺处理的肝脏中出现的部分Thy1^+细胞可以分化为+ SHs并成熟为肝细胞。Thyl+和CD44^+细胞经逆转录酶/PH处理后，可在受体肝脏中存活并增殖形成肝灶。此外，当将培养的SHs移植到经辐射/PH处理的基因大鼠肝脏中时，细胞迁移到肝小梁中，肝脏部分被供体细胞重新填充（肝移植，2006）。在透明质酸（HA； CD44配体）包被的培养皿和无血清培养基中培养的大鼠SHs可以选择性地增殖并具有肝功能（Nature Protocols, 2007），该方法可用于从患者的正常肝组织中分离人类SHs，这些组织是在知情同意的情况下通过手术切除的。人类SHs的数量在3周内增加了约100倍，细胞表现出白蛋白分泌等肝脏特征（Cell Transplant, 2008）。此外，我们发现SHs产生的卵泡抑素可以抑制成熟肝细胞分泌的激活素A的作用，从而促进大鼠SHs的增殖。另一方面，我们通过堆叠由大鼠SHs组成的二维组织成功地重建了功能性肝组织（FASEB J, 2006）。此外，我们可以长期培养正常大鼠胆道上皮细胞，并在胶原凝胶之间重建功能性胆管（Am J Pathol, 2008）。

We have reported that CD44 is specifically expressed in rat small hepatocytes and that CD44^+ hepatocytes transiently appear in injured rat livers. Using a specific antibody, we sorted CD44^+ cells from galactosamine-treated rat livers and cultured them. The phenotypes and gene expression of the cells were similar to those of cultured SHs and the ^+ cells could differentiate into hepatocytes (J Hepatol, 2006). In addition, some Thy1^+ cells that appeared in the galactosamine-treated livers could differentiate into + SHs and mature into hepatocytes. When the Thyl+ and CD44^+ cells were transplanted into livers treated with retrorsine/PH, they could survive and proliferate to form hepatic foci in the recipient livers. Furthermore, when cultured SHs were transplanted into congenic rat livers treated with radiation/PH, the cells migrated into hepatic trabecules and the liver was partially repopulated by the donor cells (Liver Transplant, 2006). Rat SHs cultured in hyaluronic acid (HA ; ligand for CD44)-coated dishes and serum-free medium could selectively proliferate with hepatic functions (Nature Protocols, 2007) and this method could be applied for the isolation of human SHs from normal liver tissues, which were surgically resected from patients with informed consent. The number of human SHs increased about 100-fold within 3 weeks and the cells showed hepatic characteristics such as albumin secretion (Cell Transplant, 2008). Furthermore, we found that the proliferation of rat SHs was enhanced by follistatin produced by SHs to inhibit the action of activin A, which is secreted by mature hepatocytes. On the other hand, we succeeded in reconstructing functional hepatic tissues by stacking up two-dimensional tissues composed of rat SHs (FASEB J, 2006). In addition, we could culture normal rat biliary epithelial cells for a long term and reconstruct functional bile ductules between collagen gels (Am J Pathol, 2008).