Isolation of hepatic progenitor cells is a promising approach for cell replacement therapy of chronic liver disease. The winged helix transcription factor Foxl1 is a marker for progenitor cells and their descendants in the mouse liver in vivo. Here, we purify progenitor cells from Foxl1-Cre; RosaYFP mice and evaluate their proliferative and differentiation potential in vitro. Treatment of Foxl1-Cre; RosaYFP mice with a 3,5-diethoxycarbonyl-1, 4-dihydrocollidine diet led to an increase of the percentage of YFP-labeled Foxl1 + cells. Clonogenic assays demonstrated that up to 3.6% of Foxl1 + cells had proliferative potential. Foxl1 + cells differentiated into cholangiocytes and hepatocytes in vitro, depending on the culture condition employed. Microarray analyses indicated that Foxl1 + cells express stem cell markers such as Prom1 as well as differentiation markers such as Ck19 and Hnf4a. Thus, the Foxl1-Cre; RosaYFP model allows for easy isolation of adult hepatic progenitor cells that can be expanded and differentiated in culture.

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Biliary atresia is a neonatal disorder characterized by aggressive fibroinflammatory obliteration of the biliary tract. Approximately 20 percent of biliary atresia patients demonstrate left-right laterality defects (syndromic biliary atresia). Cilia participate in important physiologic functions in cholangiocytes, and since some ciliopathies have been associated with both laterality defects and hepatic fibrosis, we hypothesized that patients with syndromic biliary atresia exhibit abnormalities of cholangiocyte cilia that disrupt cholangiocyte homeostasis. Nine biliary atresia specimens were studied, including pre-Kasai diagnostic biopsies (n=7) and liver explants (n=2). Five specimens were from patients with laterality defects. These were compared to normal pediatric livers as well as livers affected by primary sclerosing cholangitis, Wilson’s disease, and cardiac cirrhosis. Biopsy sections were stained with antibodies against keratin 19 (a cholangiocyte marker) and acetylated α-tubulin (a cilia marker) and were visualized by confocal microscopy. Computer-assisted relative quantification was used to compare staining of cilia within bile ducts among samples. Surprisingly, cilia in biliary atresia specimens were significantly shorter, abnormal in their orientation, and less abundant compared to normal liver and disease controls regardless of the presence of a laterality defect.ConclusionThere are significant abnormalities of cholangiocyte cilia in both syndromic and non-syndromic biliary atresia livers compared to normal livers and livers affected by other cholestatic diseases. While this may result from severe cholestasis or inflammation, it may also reflect common mechanistic pathways in different forms of biliary atresia and may have important implications for understanding the progression of the disease.

The classical form of α1-antitrypsin deficiency (ATD) is an autosomal co-dominant disorder that affects ~1 in 3000 live births and is an important genetic cause of lung and liver disease. The protein affected, α1-antitrypsin (AT), is predominantly derived from the liver and has the function of inhibiting neutrophil elastase and several other destructive neutrophil proteinases. The genetic defect is a point mutation that leads to misfolding of the mutant protein, which is referred to as α1-antitrypsin Z (ATZ). Because of its misfolding, ATZ is unable to efficiently traverse the secretory pathway. Accumulation of ATZ in the endoplasmic reticulum of liver cells has a gain-of-function proteotoxic effect on the liver, resulting in fibrosis, cirrhosis and/or hepatocellular carcinoma in some individuals. Moreover, because of reduced secretion, there is a lack of anti-proteinase activity in the lung, which allows neutrophil proteases to destroy the connective tissue matrix and cause chronic obstructive pulmonary disease (COPD) by loss of function. Wide variation in the incidence and severity of liver and lung disease among individuals with ATD has made this disease one of the most challenging of the rare genetic disorders to diagnose and treat. Other than cigarette smoking, which worsens COPD in ATD, genetic and environmental modifiers that determine this phenotypic variability are unknown. A limited number of therapeutic strategies are currently available, and liver transplantation is the only treatment for severe liver disease. Although replacement therapy with purified AT corrects the loss of anti-proteinase function, COPD progresses in a substantial number of individuals with ATD and some undergo lung transplantation. Nevertheless, advances in understanding the variability in clinical phenotype and in developing novel therapeutic concepts is beginning to address the major clinical challenges of this mysterious disorder.