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.
The biology of progenitor activation in the liver is of considerable medical and scientific interest. The powerful genetic tools available for the mouse make it an ideal model system to study this complex process involving many different cell types. However, reagents for the isolation and study of distinct hepatic subpopulations have been quite limited compared to those available for hematopoietic cells. To produce cell surface reactive reagents more specific for the oval cell response, we generated a new collection of monoclonal antibodies by immunization of Fischer rats with enzymatically dispersed nonparenchymal cells from the livers of adult mice treated with 3,5-diethoxycarbonyl-1,4-dihydrocollidine. Each of the resulting antibodies recognized a surface antigen present on a liver cell subset and permitted the viable isolation of the associated subpopulation by fluorescence-activated cell sorting. Differential activity was observed on normal liver cells and at different stages of oval cell activation, indicating potential utility for progenitor cell identification. The subdivision of liver cells using these tools should facilitate the study of the biology of ductal and periductal hepatic cell types, including progenitors. Conclusion: A new panel of surface reactive monoclonal antibodies to support investigation of the murine oval cell response has been developed. (HEPATOLOGY 2008.)
Abstract The derivation of mature functional cholangiocytes from human pluripotent stem cells (hPSCs) would provide a model for studying the pathogenesis of cholangiopathies and for developing novel therapies to treat them. Current differentiation protocols are not efficient and give rise to cholangiocytes that are not fully mature, limiting their therapeutic applications. Here, we describe a new strategy to generate functional hPSC-derived cholangiocytes that display many characteristics of mature bile duct cells including high levels of CFTR and the presence of primary cilia capable of sensing flow. With this level of maturation, these cholangiocytes are amenable for testing the efficacy of new cystic fibrosis drugs and for studying the role of cilia in cholangiocyte development and function. Transplantation studies showed that the mature cholangiocytes generate ductal structures in the liver of immunocompromised mice providing the first indication that it may be possible to develop cell-based therapies to restore bile duct function in patients with biliary disease.
In vitro modeling of human islet cells for diabetes research utilizing purified and then selectively re-aggregated various combinations of human islet cells.
The understanding of the regulation of glucagon secretion by pancreatic islet α-cells remains elusive. We aimed to develop an in vitro model for investigating the function of human α-cells under direct influence of glucose and other potential regulators.Highly purified human α-cells from islets of deceased donors were re-aggregated in the presence or absence of β-cells in culture, evaluated for glucagon secretion under various treatment conditions, and compared to that of intact human islets and non-sorted islet cell aggregates.The pure human α-cell aggregates maintained proper glucagon secretion capability at low concentrations of glucose, but failed to respond to changes in ambient glucose concentration. Addition of purified β-cells, but not the secreted factors from β-cells at low or high concentrations of glucose, partly restored the responsiveness of α-cells to glucose with regulated glucagon secretion. The EphA stimulator ephrinA5-fc failed to mimic the inhibitory effect of β-cells on glucagon secretion. Glibenclamide inhibited glucagon secretion from islets and the α- and β-mixed cell-aggregates, but not from the α-cell-only aggregates, at 2.0 mM glucose.This study validated the use of isolated and then re-aggregated human islet cells for investigating α-cell function and paracrine regulation, and demonstrated the importance of cell-to-cell contact between α- and β-cells on glucagon secretion. Loss of proper β- and α-cell physical interaction in islets likely contributes to the dysregulated glucagon secretion in diabetic patients. Re-aggregated select combinations of human islet cells provide unique platforms for studying islet cell function and regulation.
