ABSTRACT
The Mixl1 gene encodes a homeodomain transcription factor that is required for normal mesoderm and endoderm development in the mouse. We have examined the consequences of enforced Mixl1 expression during mouse embryonic stem cell (ESC) differentiation. We show that three independently derived ESC lines constitutively expressing Mixl1 (Mixl1(C) ESCs) differentiate into embryoid bodies (EBs) containing a higher proportion of E-cadherin (E-Cad)(+) cells. Our analysis also shows that this differentiation occurs at the expense of hematopoietic mesoderm differentiation, with Mixl1(C) ESCs expressing only low levels of Flk1 and failing to develop hemoglobinized cells. Immunohistochemistry and immunofluorescence studies revealed that Mixl1(C) EBs have extensive areas containing cells with an epithelial morphology that express E-Cad, FoxA2, and Sox17, consistent with enhanced endoderm formation. Luciferase reporter transfection experiments indicate that Mixl1 can transactivate the Gsc, Sox17, and E-Cad promoters, supporting the hypothesis that Mixl1 has a direct role in definitive endoderm formation. Taken together, these studies suggest that high levels of Mixl1 preferentially allocate cells to the endoderm during ESC differentiation.
Subject(s)
Cell Differentiation/physiology , Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , Endoderm/metabolism , Homeodomain Proteins/physiology , Mesoderm/cytology , Mesoderm/metabolism , Animals , BALB 3T3 Cells , Blotting, Western , Cadherins/genetics , Cadherins/metabolism , Cell Differentiation/genetics , Cell Line , Electrophoretic Mobility Shift Assay , Endoderm/cytology , Flow Cytometry , HMGB Proteins/genetics , HMGB Proteins/metabolism , Hepatocyte Nuclear Factor 3-beta/genetics , Hepatocyte Nuclear Factor 3-beta/metabolism , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Immunohistochemistry , Mice , SOXF Transcription Factors/genetics , SOXF Transcription Factors/metabolismABSTRACT
The potential for embryonic stem (ES) cells to differentiate into cells with a distal lung epithelial phenotype has been demonstrated using different in vitro culture methods. Three separate protocols are described here that utilize both murine and human ES cells. The distal lung epithelial phenotype is induced through the use of embryonic distal lung mesenchyme in coculture systems with differentiating embryoid bodies or the use of soluble factors in defined media to maximize definitive endoderm formation and select and maintain the desired phenotype. Phenotypic analysis is demonstrated using immunocytochemistry and SP-C promoter-eGFP reporter gene expression in transgenic ES cells. These methods provide an increased efficiency of distal lung epithelial derivation from ES cells and, therefore, they provide the foundation for the development of a cell replacement product to treat chronic lung disease or a useful in vitro model for the study of lung disease and development.