Cellular and Molecular Biology of the Hepatic Stem Cell Compartment

我们建立了一个从小鼠胚胎干细胞中分化、扩增和分离肝祖细胞的有效系统，并评估了它们在损伤肝脏中的再生能力。利用转染了由白蛋白（ALB）增强子/启动子调控的绿色荧光蛋白（GFP）报告基因的小鼠胚胎干细胞，我们发现无血清的化学定义培养基支持胚状体（EBs）的形成和肝系细胞在缺乏外源生长因子或饲养细胞层的情况下的分化。在EB培养7天后，第一批表达ALB的GFP阳性细胞在跳动的肌细胞附近被检测到。GFP阳性细胞数量增加，获得肝细胞样形态和肝细胞特异性标记物（即ALB， AAT， TO和G6P），并且在28天时占EB生长产物分离细胞的30%以上。facs纯化的GFP细胞在移植到MUP-uPA/SCID小鼠体内后，发育为无细胞融合的功能性肝细胞，并参与了病变肝脏的修复。胚胎干细胞来源的肝细胞对正常生长调节有反应，在ccl4诱导的肝损伤的额外生长刺激后，其增殖速度与宿主肝细胞相同。移植的GFP阳性细胞也分化为胆道上皮细胞。综上所述，胚胎干细胞可以在体外产生高度富集的肝细胞前体，从而实现有效的细胞替代治疗。2-乙酰氨基芴/部分肝切除术（AAF/PH）模型被广泛用于诱导大鼠肝脏卵圆细胞/祖细胞增殖。我们使用该模型研究了原代肝细胞有丝分裂原三碘甲状腺原氨酸（T3）对卵形/祖细胞募集的肝脏再生的影响。PH后第5天给予三碘甲状腺原氨酸（T3）不能诱导肝细胞有丝分裂反应。然而，卵形细胞的反应是加速增殖，随后迅速分化为小肝细胞。在加速分化过程中，卵形细胞特异性OV-6和α胎蛋白（AFP）的表达丢失，在新形成的小的嗜碱性肝细胞中，肝细胞核因子(HNF)-4上调。分化的卵形细胞也开始表达肝细胞特异性连接素（Cx） 32、整合素1和细胞色素p450，形成cd26阳性胆管。采用实时荧光定量PCR检测小肝细胞白蛋白、酪氨酸转氨酶（TAT）、色氨酸2,3-双加氧酶（TO2）等肝细胞特异性mrna的上调。此外，肝功能测试的改善与分化的加速同时发生。在该模型中，卵形细胞在服药后第7天出现肝细胞转化，未给予T3治疗的小鼠在服药后第11 -12天出现第一批小肝细胞。我们得出结论，原代肝细胞有丝分裂原T3在体内加速肝祖细胞向肝细胞的分化，可能具有治疗潜力。HGF/Met信号系统对肝脏发育、体内平衡和功能至关重要。在本研究中，我们利用之前在我们实验室产生的肝脏特异性Met条件敲除小鼠来解决Met在成人肝祖细胞（卵形细胞）生物学中的作用。为此，我们从ddc处理的Metflx/flx小鼠中分离卵形细胞，然后利用病毒介导的Cre-loxP重组建立卵形细胞衍生细胞系，这些细胞系携带功能性（Metflx/flx）或非功能性（Met-/-） Met基因。缺乏Met激酶活性的卵形细胞在表型上与Metflx/flx对照相似，但既没有Met磷酸化，也没有Met下游靶点的激活。此外，Met-/-细胞对HGF的增殖刺激是难以耐受的。虽然Met-/-和Metflx/flx细胞在10%的血清中增殖率相似，但Met缺乏的细胞表现出细胞活力下降，在血清饥饿或促凋亡细胞因子tgf - β的刺激下更容易凋亡。HGF治疗减少了Metflx/flx中tgf - β介导的细胞死亡，但没有减少Met-/-细胞的死亡。重要的是，Metflx/flx和Met-/-细胞均组成性表达HGF，血清饥饿卵形细胞的条件培养基在Metflx/flx细胞中表现出抗凋亡活性。此外，血清饥饿的Metflx/flx细胞显示Met酪氨酸激酶持续激活，表明HGF/Met自分泌调节。综上所述，这些数据表明Met通过自分泌机制在卵圆细胞存活中发挥了关键的功能作用

We have established an efficient system for differentiation, expansion and isolation of hepatic progenitor cells from mouse embryonic stem (ES) cells and evaluated their capacity to repopulate injured liver. Using mouse ES cells transfected with the green fluorescent protein (GFP) reporter gene regulated by albumin (ALB) enhancer/promoter, we found that a serum-free chemically defined medium supports formation of embryoid bodies (EBs) and differentiation of hepatic lineage cells in the absence of exogenous growth factors or feeder cell layers. The first GFP positive cells expressing ALB were detected in close proximity to beating myocytes after 7 days of EB cultures. GFP positive cells increased in number, acquired hepatocyte-like morphology and hepatocyte specific markers (i.e., ALB, AAT, TO, and G6P), and by 28 days represented more than 30% of cells isolated from EB outgrowths. The FACS-purified GFP cells developed into functional hepatocytes without evidence of cell fusion and participated in the repairing of diseased liver when transplanted into MUP-uPA/SCID mice. The ES cell-derived hepatocytes were responsive to normal growth regulation and proliferated at the same rate as the host hepatocytes after an additional growth stimulus from CCl4-induced liver injury. The transplanted GFP positive cells also differentiated into biliary epithelial cells. In conclusion, a highly enriched population of committed hepatocyte precursors can be generated from ES cells in vitro for effective cell replacement therapy. The 2-acetaminofluorene/partial hepatectomy (AAF/PH) model is widely used to induce oval/progenitor cell proliferation in the rat liver. We have used this model to study the impact of a primary hepatocyte mitogen, triiodothyronine (T3) on the liver regenerating by the recruitment of oval/progenitor cells. Administration of triiodothyronine (T3) on the 5th day after the PH can not induce mitogenic reaction in the hepatocytes. However, the oval cells respond by accelerated proliferation, which is followed by rapid differentiation into small hepatocytes. During the accelerated differentiation the oval cell specific OV-6 and alpha-fetoprotein (AFP) expression is lost, hepatocyte nuclear factor (HNF)-4 is upregulated in newly formed small, basophilic hepatocytes. The differentiating oval cells also start to express hepatocyte specific connexin (Cx) 32, 1 integrin and cytochrom P450s, and form CD 26 positive bile canaliculi. The upregulation of hepatocyte specific mRNAs e.g. albumin, , tyrosine aminotransferase ( TAT) and tryptophan 2,3-dioxygenase (TO2) was detected by by quantitative real time PCR in microdissected small hepatocytes. Furthermore, improved liver function tests happens concurrently with the accelerated differentiation. The hepatocytic conversion of oval cells occurs on the 7th day after the Phx in this model while the first small hepatocytes appear on the 11th-12th day post Phx without T3 treatment. We conclude that administration of the primary hepatocyte mitogen T3 accelerates the differentiation of hepatic progenitor cells into hepatocytes in vivo, and that may have therapeutic potential. The HGF/Met signaling system is essential for liver development, homeostasis and function. In this study, we took advantage of the liver specific Met conditional knockout mouse generated in our laboratory previously to address the role of Met in adult liver progenitor cell (oval cell) biology. For this purpose, we isolated oval cells from DDC-treated Metflx/flx mice, and then established oval cell derived cell lines that carried either functional (Metflx/flx) or non-functional (Met-/-) met gene using virus-mediated Cre-loxP recombination. Oval cells lacking Met kinase activity were phenotypically similar to Metflx/flx controls but displayed neither Met phosphorylation, nor activation of Met downstream targets. In addition, Met-/- cells were refractory to HGF stimulation of proliferation. Although Met-/- and Metflx/flx cells proliferated at a similar rate in the presence of 10% serum, Met deficient cells demonstrated decreased cell viability, and were more prone to apoptosis when challenged with serum starvation or the pro-apoptotic cytokine TGF-beta. Treatment with HGF reduced the TGF-Beta-mediated cell death in Metflx/flx but not Met-/- cells. Importantly, both Metflx/flx and Met-/- cells constitutively expressed HGF, and conditioned medium from serum starved oval cells exhibited antiapoptotic activity in Metflx/flx cells. Furthermore, serum-starved Metflx/flx cells showed persistent activation of Met tyrosine kinase suggesting HGF/Met autocrine regulation. In conclusion, these data assign a critical functional role for Met in oval cell survival through an autocrine mechanism