Basic research on treatment of liver failure and prevention against liver fibrosis by transplantation of MAPC-like stem cells established from bone marrow

肝外来源的肝细胞或肝细胞样细胞移植是治疗急、慢性肝衰竭的一种有前景的策略。我们研究了从骨髓细胞诱导的肝细胞样细胞用于此目的的可能效用。我们从大鼠骨髓中建立了rBM 25/S3细胞系。细胞快速生长（倍增时间，约24小时），在300天的时间内至少300 PDL，细胞性质没有任何明显变化，保持正常的二倍体核型。细胞表达CD 29、CD 44、CD 49 b、CD 90、波形蛋白和纤连蛋白，但不表达CD 45，表明它们是间充质细胞来源的。当在具有HGF和FGF-4的Matrigel上铺板时，细胞有效地分化成表达白蛋白、CYP 1A 1、CYP 1A 2、G6 β、TO、达特、HNF 1 α和HNF 4 α的肝细胞样细胞。当在相同的培养基中培养，但在胶原基质上，细胞显着表现出成脂特性，如脂滴在细胞质中的进行性积累和脂联素，瘦素和Adiponin的表达。我们脾内移植的三种类型的细胞到大鼠90%肝切除术前诱导致命的肝衰竭，或大鼠之前和之后开始的CCl_4治疗诱导肝纤维化。脾内移植肝细胞样细胞挽救了15只大鼠中的5只，致命的肝功能衰竭。此外，移植未分化的rBM 25/S3细胞最有效地抑制肝纤维化，其次是脂肪细胞和肝细胞。MMP-2和MMP-9在未分化的rBM 25/S3细胞中的表达也最高。总之，骨髓源性克隆干细胞系（rBM 25/S3）适合于治疗肝纤维化，肝源性分化衍生物是有效的拯救肝衰竭。因此，我们将进一步研究同一克隆来源的不同细胞类型的联合移植治疗急性和慢性肝功能衰竭的效果。

Transplantation of hepatocytes or hepatocyte-like cells of extrahepatic origin is a promising strategy for treatment of acute and chronic liver failure. We examined possible utility of hepatocyte-like cells induced from bone marrow cells for such a purpose. We established a clonal cell line rBM25/S3 from rat bone marrow. The cells grew rapidly (doubling time,〜24 hours) without any appreciable changes in cell properties for at least 300 PDL over a period of 300 days, keeping normal diploid karyotype. The cells expressed CD29, CD44, CD49b, CD90, vimentin and fibronectin but not CD45, indicating that they are of mesenchymal cell origin. When plated on Matrigel with HGF and FGF-4, the cells efficiently differentiated into hepatocyte-like cells that expressed albumin, CYP1A1, CYP1A2, G6Pase, TO, TAT, HNFlα, and HNF4α. When cultivated in the same medium but on a collagen matrix, the cells prominently exhibited adipogenic properties, such as progressive accumulation of lipid droplets in the cytoplasm and expression of adiponectin, leptin and resistin.We intrasplenically transplanted the three types of cells into rats before 90% hepatectomy to induce fatal liver failure, or rats before and after initiation of CC1_4 treatment to induce liver fibrosis. Intrasplenic transplantation of hepatocyte-like cells rescued 5 out of 15 rats with,fatal liver failure. Furthermore, transplantation of undifferentiated rBM25/S3 cells most effectively suppressed liver fibrosis, followed by those of the adipogenic cells and the hepatogenic cells. Expression of MMP-2 and MMP-9 was also highest in undifferentiated rBM25/S3 cells.In conclusion, the bone marrow-derived clonal stem cell line (rBM25/S3) is suitable to treat liver fibrosis and the hepatogenically-differentiated derivative is potent to rescue liver failure. So we will further examine effects of combined transplantation of the different cell types of the same clonal origin on treatment of acute and chronic liver failure.