The liver is composed of diverse cell types that arise from various embryonic origins. Among those cell types, hepatocytes, the parenchymal cells performing the major functions of the organ, and cholangiocytes, lining the bile ducts, are the two epithelial cell lineages. Homeostasis of the adult liver and also regeneration after partial hepatectomy can be achieved by cell division of mature hepatocytes and cholangiocytes, whereas in damaged liver with defects in hepatocyte proliferation, adult liver progenitor cells (LPCs) emerge usually in the periportal area and have the potential to differentiate into both hepatocytes and cholangiocytes. However, whether LPCs indeed engage in liver regeneration has not been demonstrated.

The fibroblast growth factor (FGF) family members have diverse roles in physiological and pathological conditions both during development and in adulthood. Since little is known about the role of FGF signaling in LPC activation and/or liver regeneration, I examined by PCR analysis the expression of FGFs in the livers of mice fed a 3,5-Diethoxycarbonyl-1,4-Dihydrocollidine (DDC)-containing diet. I found the expression of Fgf7 to increase significantly during the time course of DDC-induced liver damage, along with that of Epcam and Krt19, encoding the LPC/cholangiocyte markers epithelial cell adhesion molecule (EpCAM) and cytokeratin 19 (CK19), respectively. The results of immunostaining showed that FGF7 was detected only in the smooth muscle of the arteries in the normal liver. On the other hand, there was a marked increase of FGF7 expression in the vicinity of LPCs under conditions of hepatic injury. To address the physiogical relevance of FGF7 expression, I used Fgf7 knockout (KO) mice. Fgf7 KO mice exhibit normal growth and mild phenotypes, and show no liver phenotype during development or in adulthood. In order to analyze the LPC response in Fgf7 KO mice, adult littermates of wild type (WT) and KO mice were fed a normal or DDC-containing diet. I measured the degree of LPC activation by immunostaining CK19, a well-established LPC/cholangiocyte marker, and confirmed that CK19+ LPC numbers were increased by DDC in the WT liver. However, the LPC response was almost completely suppressed in Fgf7 KO mice at 2 weeks and later on (Figure 1). The KO mice were highly sensitive to DDC and had a low survival rate, whereas the WT mice were resistant to the hepatotoxin-induced liver injury. This study demonstrates, for the first time, that the extent of LPC activation via FGF7 signaling significantly affects the mortality and the regenerative capacity of the liver.

I then examined the relationship between FGF7 and LPCs in other models of liver injury. First, I checked the activation of LPCs and expression of FGF7 in liver-specific Tak1-deficient (Tak1-LKO) mice. Those mice have been reported as a liver injury model that eventually show cholestasis and carcinogenesis. LPC activation was observed in 8-week-old Tak1-LKO mice. The expression of Fgf7 was augmented with the LPC activation. Second, the LPC response after ligation of the common bile duct was significantly inhibited in Fgf7 KO mice. These findings suggest that FGF7 is necessary for LPC activation in vivo, and its expression and function may counter liver dysfunction.

I next performed gain-of-function experiments to further explore the function of FGF7 in regulating the LPC response. To begin with, I examined the effect of FGF7 on LPCs in vitro. I found that a recombinant FGF7 stimulated the proliferation of HSCE5, a cell line derived from EpCAM+ LPCs of adult mice, in a dose-dependent manner. To reveal the effect of FGF7 in vivo, I examined Alfp-Cre ; Rosa26-loxP-STOP-loxP-rtTA; tetO-CMV-FGF7 triple transgenic (Tg) mice in which liver-specific FGF7 overexpression could be achieved upon doxycycline (Dox) treatment. A significant increase in CK19+ LPC-like cell numbers was observed in the liver of the triple Tg mice, compared to control Alfp-Cre Rosa26-loxP-STOP-loxP-rtTA double Tg mice. As an alternative system to achieve ectopic gene expressions in the adult mouse liver, I also exploited hydrodynamic tail vein injection (HTVi)-mediated gene transfer. It has been established that high levels of foreign gene expression in 10-40% of mouse hepatocytes can be achieved by this method with little liver damage. Indeed, long-term expression of FGF7 was accomplished and continued for several weeks. Notably, the mice with FGF7 overexpression by HTVi showed similar phenotypes to epidermis-specific FGF7 Tg mice, like a wet undercoat caused by excessive salivary secretions and retardation of weight gain, suggesting that the functional protein was adequately produced and secreted. I found that an EpCAM+ LPC-like population was induced by overexpression of FGF7 via HTVi. While the LPC-specific marker Trop2 was not detected in the control, 22-29% of the EpCAM+ cells were Trop2-positive in the liver where FGF7 was overexpressed. The EpCAM+ cells induced by overexpression of FGF7 also expressed other well-known LPC markers, CK19 and A6. Therefore, it seems reasonable to conclude that FGF7 alone is sufficient to generate an LPC-like population.

The severity of the liver damage induced by DDC was relieved by an excess of FGF7, according to blood tests. Though serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were markedly elevated after the administration of DDC in both control and FGF7-overexpressing mice, increases in alkaline phosphatase (ALP) and total bilirubin (TBIL) levels were greatly reduced by the HTVi of Fgf7. These results indicate that bile duct obstruction was alleviated by FGF7 overexpression under conditions where hepatocytes were persistently destroyed by DDC. Therefore, I considered that FGF7 could accelerate the functional recovery of hepatocytes and/or cholangiocytes through LPC activation.

It has been proposed that FGF7 is a mesenchymal cell-derived paracrine cytokine, which has specific mitogenic effects on epithelial cells in many organs. As LPCs are regarded as epithelial-type cells, I hypothesized that FGF7 was produced by some sort of mesenchymal cell. Immunostaining of the liver sections with several mesenchymal cell markers showed that Thy1+ cells appeared in close proximity to LPCs after liver damage, as described in rats and humans. An established marker for fibroblastic cells, Elastin, and a stellate cell marker, Desmin, were partially expressed in Thy1+ mesenchymal cells. Quantitative analysis of the Thy1 and CK19 immunostaining revealed that the expansion of Thy1+ cells occurred prior to LPC activation. Intriguingly, the Thy1+ cells were co-stained with FGF7 in the injured liver. I then analyzed the expression pattern of the FGF7 receptor, fibroblast growth factor receptor 2 isoform IIIb (FGFR2b). Among the non-parenchymal cell (NPC) populations, EpCAM+ LPCs and EpCAM- cells isolated from DDC-treated livers were subjected to immunostaining with an IIIb isoform-specific anti-FGFR2 antibody. I found that only EpCAM+ cells expressed FGFR2b. To further confirm the FGF7-producing cells and receiving cells, I performed a quantitative PCR analysis using specific cell populations. Hepatocyte, NPC, EpCAM+ LPC, Thy1+ CD45- mesenchymal cell (Thy1+ MC), Thy1+ CD45+ cell (T-cell) and Thy1- CD45+ cell (blood cell) fractions were isolated from the livers of mice fed DDC. I checked for adequate cell separation by the specific expression of each marker. As expected, Fgf7 and Fgfr2 isoform IIIb were detected in Thy1+ MC and LPC fractions, respectively. Since these FGF7-producing Thy1+ CD45- cells strongly expressed Elastin (Eln), a fibroblastic cell marker, they are considered to be a mesenchymal cell population and distinct from T-cell populations. Fibroblast growth factor binding protein 1 (FGFBP1) is a soluble protein that can bind a subset of FGFs, including FGF7, and enhance their activities. Previous studies on skin and renal tube regeneration have shown FGFBP1 to be expressed in epithelial cells rather than mesenchymal cells and to be a target of FGF7 signaling. LPC-specific expression of Fgfbp1 further strengthened the notion that LPCs are the primary target of FGF7 signaling from Thy1+ cells. Interestingly, a quantitative analysis of the Thy1+ area in Fgf7 KO mice revealed little change when compared to the WT control in both normal and damaged liver. In other words, Thy1+ cells were capable of increasing in number in response to liver damage irrespective of FGF7 function, while LPCs failed to proliferate due to a lack of FGF7 produced by Thy1+ cells. In general, tissue stem cells are supported and regulated by their surrounding microenvironment, or the stem cell niche. While several molecules that participate in the LPC response have been reported, the possible involvement of niche signals has not. In this study, I have identified FGF7 as a key factor for the LPC response and that Thy1+ periportal mesenchymal cells form the niche that produces FGF7 (Figure 2).

While it has long been documented that LPCs appear and proliferate in injured and cancerous livers, whether LPCs indeed engage in regeneration has not been clear. My data, based on loss- and gain-of-function experiments with FGF7, demonstrate for the first time that the level of LPC activation correlates with resilience and survival in cases of severe liver injury, and strongly suggest that LPCs directly contribute to liver regeneration.

Figure1.LPC response upon DDC administration is suppressed in Fgf7 KO mice.

Figure2.Schematic model.

本論文は5つのChapterからなる。Chapter1は序論、Chapter2は材料と方法Chapter3は実験結果、Chapter4は考察、Chapter5は結論について記述されている。

申請者の所属する研究室では成体肝幹/前駆細胞(liverprogenitorcell:LPC)に着目し、その性状を解析するために、表面抗原の同定、セルソーターを用いた分離法の確立、in vitroにおける分化能の評価等を行ってきた。申請者はこれらの評価系を用いて解析を進め、LPCの出現と増殖に必須な増殖因子としてfibroblastgrowthfactor7(FGF7)を同定し、さらに遺伝学的手法を用いた解析によりLPCが肝障害下で再生へ寄与していることを示した。

重篤な障害を受けた肝臓では、LPCと呼ばれる特殊な細胞が出現することが知られている。LPCは未分化マーカーを発現することから成体肝幹/前駆細胞とみなされているが、実際に肝再生に寄与しているのか明確ではなく、またその発生機構や動態については不明な点が多く残されていた。本論文では、LPCを制御する分子メカニズムを明らかにし、さらにLPCの肝再生への関与を検証することを目指し、詳細に解析している。

申請者の所属する研究室ではLPC特異的マーカーとしてTrop2を「司定している。肺の発生過程においてはFGF10がTrop2の発現を誘導することが知られており、申請者はLPCの制御にFGFファミリーの関与する可能性を考え、これを検討した。その結果、3,5-Diethoxycarbonyl-1,4-Dihydrocollidine(DDC)投与等のマウス肝障害モデルにおいて、LPCの出現に伴い、その周囲でFGF7の発現が強く誘導されることを見いだした。FGF7欠損マウスは、通常の飼育条件下では目立った表現型が見られず、肝臓も正常に発生する。しかしながら、FGF7欠損マウスでは肝障害に伴うLPCの誘導が顕著に抑制されており、長期間のDDC食餌投与により肝障害が増悪化し、致死率が有意に上昇した。この結果は、FGF7がLPCの誘導に必須の因子であること、さらにFGF7を介したLPCが肝臓の再生に積極的に寄与することを示すものと考えられる。

一方で、FGF7の肝臓での強制発現系を用いた解析から、FGF7により正常肝でもLPC様の細胞が出現することが判明した。FGF7の過剰発現により出現するLPC様の細胞は、その一部がLPC特異的マ-カ-Trop2を発現することから、この細胞は肝障害時に現れるLPCに近い性質を持っていると推測された。さらに、血液生化学検査により、FGF7の過剰発現はDDC投与による肝障害を軽減させることが判明した。この結果はFGF7の発現あるいは投与によりLPCを誘導あるいは活性化することで、肝障害の軽減化、肝機能の回復を促すことができる可能性を示すものである。

一般的に、組織幹/前駆細胞はニッチと呼ばれる微小環境によって制御される。一ヒ記の結果はFGF7がLPCのニッチシグナルであることを示しており、申請者はニッチを形成する細胞群を同定することを試みた。免疫染色の結果から、LPCの周囲にはThy1陽性の間葉系細胞が存在し、障害に応答してLPCの出現に先立って増殖することが見出された。さらに、フローサイトメーターを用いて肝臓構成細胞を分画して遺伝子発現解析を行ったところ、Thy1陽性の間葉系細胞がFGF7を産生し、LPCはFGF7の特異的受容体であるFGFR2bを発現していることが明らかとなった。したがって、Thy1陽性細胞は]」PCのニッチを形成していると考えられる。

以上の結果から、障害肝においてThy1陽性細胞がFGF7の産生を介してLPCを誘導すること、誘導されたLPCは肝障害からの回復・再生に重要な役割を担うことが強く示唆された。本研究は、LPCの制御機構の分子細胞生物学的実体を明らかにし、FGF7の臨床応用の可能性を示したという点で意義深い。この成果は、肝臓、幹細胞、再生医療の研究分野の進展に寄与することが大いに期待される。

なお、本論文のChapter3は伊藤暢、宮島篤との共同研究であるが、申請者が主体となって実験及び考察を行ったものであり、申請者の寄与が十分であると判断する。よって、博士(理学)の学位を授与できると認める。