人成纤维细胞转分化为肝干细胞的研究

Recent studies indicated that cell transplantation is expected to become an effective treatment for end-stage liver disease. Repopulated hepatocytes in the host liver proliferated slowly, so it is difficult to achieve therapeutic liver repopulation after hepatocytes transplantation. While hepatic stem cells possess the capacity of self-renewal and the potential of bidirectional differentiation into both hepatocytes and cholangiocytes. The hepatic stem cells transplantation was considered to be the most promising method of treatment of end-stage liver disease. But the number of the hepatic stem cells in the liver is very few. Simultaneously the hepatic stem cells were very difficult to separate. So the source of the hepatic stem cells is an urgent problem to be solved. Lineage reprogramming achieved the direct transdifferentiation between different cell types and avoided the inefficiency of hepatic differentiation and the potential tumorigenic risk of ES/iPS cells. Human fibroblasts were induced to directly transdifferentiate into induced hepatic stem cells (ihHepSCs) by lineage reprogramming in this study. Then the characteristics of the ihHepSCs were identified in vitro. ihHepSCs were transplanted into immunodeficient hepatocellular injury model (Fah knockout mice) and DDC-induced bile duct injury model mice, respectively, to evaluate the therapeutic effects on these liver damage models and the ability to differentiate into mature hepatocytes and cholangiocytes in vivo. These results lay the foundation for the study of patient costumed hepatic stem cells therapy for end-stage liver disease.

研究表明，肝细胞移植有望成为终末期肝病的有效治疗手段。然而，成熟肝细胞移植后在宿主肝脏内增殖缓慢，难以实现治疗性肝脏再殖。肝干细胞具有自我更新能力和向肝细胞和胆管细胞双向分化的潜能，利用肝干细胞移植治疗终末期肝病被认为是最有前途的治疗方案之一，但正常肝脏内肝干细胞的数量稀少且难以分离纯化，肝干细胞的来源成为急需解决的重要问题。谱系重编程实现了不同成体细胞间的直接转分化，避免了ES/iPS细胞肝向诱导分化的低效及潜在的致瘤风险。本研究通过谱系重编程的技术实现人成纤维细胞直接转分化为诱导型肝干细胞（ihHepSCs），开展ihHepSCs的肝干细胞特性鉴定，并将ihHepSCs移植到肝细胞损伤模型FAH基因剔除小鼠和DDC诱导的胆管损伤模型小鼠，评估ihHepSCs在体内分化为成熟肝细胞和胆管细胞的能力以及对肝脏疾病模型的治疗作用，从而为终末期肝病的个性化肝干细胞治疗的研究奠定理论和技术基础。

细胞移植是治疗终末期肝病的有效治疗方法之一，但供体细胞的缺乏限制了其广泛应用。谱系重编程可实现不同细胞间的直接转分化，在前期的研究中，通过谱系重编程的方法，我们将胚胎成纤维细胞直接转分化成为具有自我更新能力和双向分化潜能的诱导型肝干细胞（iHepSCs）。.本研究在前期建立的小鼠肝干细胞谱系重编程的技术体系上，通过逐渐递减因子的方法从8个候选因子中筛选出将人成纤维细胞转分化肝干细胞的关键三因子（ONECUT1，FOXA3和HNF1A）。谱系重编程方法获得的肝干细胞共同表达肝细胞特异性标志物Alb和胆管上皮细胞标志物Ck19，幼稚肝细胞的标志物AFP以及肝干细胞异性标志物（CD133、Epcam和LGR5等），同时转分化获得的CD133阳性的肝干细胞具有肝干细胞双向分化的能力和肝干细胞特征性的代谢谱。.Fibroblasts经谱系重编程为肝干细胞后获得了自我更新的能力。经基因表达谱分析发现iHepSCs中的Pitx2基因的表达水平明显高于Fibroblasts，通过shRNA基因干扰的方法将iHepSCs中的Pitx2基因的表达水平knockdown后，发现iHepSCs自我更新能力显著降低，Pitx2主要通过阻滞细胞周期中G1/S的转变降低iHepSCs更新能力。此外，Pitx2基因knockdown后，iHepSCs向肝细胞和胆管上皮细胞的分化效率和分化过程都明显提升。.诱导iHepSCs定向分化为成熟肝细胞，是解决细胞治疗肝脏疾病中所面临的成熟肝细胞来源短缺的方法之一。利用小分子化合物抑制iHepSCs的TGF-β和Notch信号通路的同时，采用HGF、OSM 和Dex等促肝向分化因子在无血清培养条件下将iHepSCs定向诱导分化为具有与成熟肝细胞的形态特征和功能的肝细胞样细胞（Hep-iHepSCs）。Hep-iHepSCs移植至慢性肝损伤模型Fah-/-小鼠后，Hep-iHepSCs的再殖效率与对损伤肝脏肝功能的恢复都明显优于于未分化的iHepSCs。提示了分化成熟的肝细胞较未分化的肝干细胞更适合慢性肝损伤的移植治疗。.该研究为人肝干细胞和成熟肝细胞的获得提供了一种新的思路，即通过谱系重编程的方法将成纤维细胞转分化为具有增殖能力的肝干细胞，随后再诱导肝干细胞分化为成熟肝细胞，从而解决细胞移植治疗和基于肝细胞药物筛选过程中肝细胞来源紧缺的瓶颈问题。