Expression of markers of endometriosis in differentiating mouse embryonic stem cells

ObjectiveDevelop an in vitro model of human disease for endometriosisDesignBasic scienceMaterials and MethodsG4 mouse embryonic stem cells (mESCs) were cultured andallowed to differentiate for 21 days as colonies or as suspended embryoid bodies (EBs). Differentiation of G4 ESCs was induced by transferring the cells to mouse embryonic feeder cell culture media (Dulbecco’s modified Eagle’s Medium (DMEM) with 10% fetal bovine serum (FBS)) for 3 weeks. G4 ESCs were also differentiated with differentiation media (DMEM, FBS, Bone morphogenetic protein (BMP)-4 50ng/ul and Activin-A 20ng/ul). Post-differentiation analysis profiles of the mESCs and EBs were assessed for the endometrium and endometriosis markers CD9/CD13 by immunocytochemistry and transcriptional analysis using reverse and quantitative PCR. Differentiated CD9+CD13+ were then prepared for sorting using FACS for further characterization.ResultsColonies of mESCs showed progressive differentiation of CD9 immunoreactive cells over a 3 week period (26.6%, 48.9%,and 76.4%, weeks 1-3, respectively), while populations of differentiated CD13 cells appeared relatively constant over this same duration (40.6%, 47.0% and 47.4%). 40% of EBs displayed CD9 immunoreactivity with 61.2% also immunolableing for CD13. These observations were confirmed using quantitative PCR analyses. Additional markers of endometrium (CD146, PGDF-b) were also identified in these differentiating cultures.ConclusionsThe results from this study demonstrate the generation of presumptive endometrial precursors in differentiating mouse ESCs. The ability to produce CD9+CD13+ cells in vitro, isolate the same by FACS and continue their in vitro growth shows the utility of ESCs for preclinical modeling of a human disease, endometriosis. Furthermore this system affords the unique opportunity to investigate novel therapeutic approaches for the management of this debilitating disease using a stem cell disease platform. ObjectiveDevelop an in vitro model of human disease for endometriosis Develop an in vitro model of human disease for endometriosis DesignBasic science Basic science Materials and MethodsG4 mouse embryonic stem cells (mESCs) were cultured andallowed to differentiate for 21 days as colonies or as suspended embryoid bodies (EBs). Differentiation of G4 ESCs was induced by transferring the cells to mouse embryonic feeder cell culture media (Dulbecco’s modified Eagle’s Medium (DMEM) with 10% fetal bovine serum (FBS)) for 3 weeks. G4 ESCs were also differentiated with differentiation media (DMEM, FBS, Bone morphogenetic protein (BMP)-4 50ng/ul and Activin-A 20ng/ul). Post-differentiation analysis profiles of the mESCs and EBs were assessed for the endometrium and endometriosis markers CD9/CD13 by immunocytochemistry and transcriptional analysis using reverse and quantitative PCR. Differentiated CD9+CD13+ were then prepared for sorting using FACS for further characterization. G4 mouse embryonic stem cells (mESCs) were cultured andallowed to differentiate for 21 days as colonies or as suspended embryoid bodies (EBs). Differentiation of G4 ESCs was induced by transferring the cells to mouse embryonic feeder cell culture media (Dulbecco’s modified Eagle’s Medium (DMEM) with 10% fetal bovine serum (FBS)) for 3 weeks. G4 ESCs were also differentiated with differentiation media (DMEM, FBS, Bone morphogenetic protein (BMP)-4 50ng/ul and Activin-A 20ng/ul). Post-differentiation analysis profiles of the mESCs and EBs were assessed for the endometrium and endometriosis markers CD9/CD13 by immunocytochemistry and transcriptional analysis using reverse and quantitative PCR. Differentiated CD9+CD13+ were then prepared for sorting using FACS for further characterization. ResultsColonies of mESCs showed progressive differentiation of CD9 immunoreactive cells over a 3 week period (26.6%, 48.9%,and 76.4%, weeks 1-3, respectively), while populations of differentiated CD13 cells appeared relatively constant over this same duration (40.6%, 47.0% and 47.4%). 40% of EBs displayed CD9 immunoreactivity with 61.2% also immunolableing for CD13. These observations were confirmed using quantitative PCR analyses. Additional markers of endometrium (CD146, PGDF-b) were also identified in these differentiating cultures. Colonies of mESCs showed progressive differentiation of CD9 immunoreactive cells over a 3 week period (26.6%, 48.9%,and 76.4%, weeks 1-3, respectively), while populations of differentiated CD13 cells appeared relatively constant over this same duration (40.6%, 47.0% and 47.4%). 40% of EBs displayed CD9 immunoreactivity with 61.2% also immunolableing for CD13. These observations were confirmed using quantitative PCR analyses. Additional markers of endometrium (CD146, PGDF-b) were also identified in these differentiating cultures. ConclusionsThe results from this study demonstrate the generation of presumptive endometrial precursors in differentiating mouse ESCs. The ability to produce CD9+CD13+ cells in vitro, isolate the same by FACS and continue their in vitro growth shows the utility of ESCs for preclinical modeling of a human disease, endometriosis. Furthermore this system affords the unique opportunity to investigate novel therapeutic approaches for the management of this debilitating disease using a stem cell disease platform. The results from this study demonstrate the generation of presumptive endometrial precursors in differentiating mouse ESCs. The ability to produce CD9+CD13+ cells in vitro, isolate the same by FACS and continue their in vitro growth shows the utility of ESCs for preclinical modeling of a human disease, endometriosis. Furthermore this system affords the unique opportunity to investigate novel therapeutic approaches for the management of this debilitating disease using a stem cell disease platform.