Abstract
We have studied the in vitro differentiation of murine embryonic stem cells (ES cells) towards erythropoiesis and expression of genes during this process. It has been reported that dexamethasone directs ES cells towards erythrocytic differentiation but the mechanism of gene regulation induced by dexamethasone is not well understood. We hypothesized that dexamethasone induces upregulation of erythropoietic genes such as GATA-1, FLK-1, EPO-R and directs ES cells towards erythropoietic differentiation. Murine ES cells (129 CCE) obtained from Dr. Nagy laboratory, Canada (Nagy et al., Histochem Cell Biol., 2001; 115:49–58) were subjected to the in vitro primary hematopoietic differentiation media containing methylcellulose, IMDM, IL -3, IL-6 and SCF (stem cell factor) without LIF (leukemia inhibitory factor) to promote embryoid body (EB) formation. Total RNA was collected on day 3, 5 and 9 EBs for gene expression studies using RT-PCR. On day 9 EBs were subjected to secondary differentiation using three different cytokines and growth factors combination 1) SCF, EPO, dexamethasone, IGF, 2) SCF, IL-3, IL-6, TPO, 3) SCF IL-3, IL-6, TPO, EPO. Total RNA from day12 of secondary differentiated ES cells was collected to study cytokines and growth factors dependent erythrocytic differentiation and gene regulation, using RT-PCR. Our results demonstrate upregulation of Gata-1, Flk-1, HoxB-4, Epo-R and globin genes (α-globin, BH-1 globin, β-major globin, e -globin and z-globin) in the 9 days old EBs, whereas, RNA collected from 5 days old EBs showed expression of HoxB-4, e-globin, γ-globin, BH1-globin and FLK-1. Three days old EBs showed only HoxB-4 and FLK-1 gene expression and lack of expression of globin genes, indicating that erythtropoiesis-specific genes activate later. Gene expression studies of RNA collected from secondary differentiated ES cells and media containing dexamethasone showed downregulation of GATA-3 and upregulation of GATA-1, Flk-1 and Epo-R in comparison to the two other cytokines and growth factors media combination. These results confirm our hypothesis that dexamethasome induces erythropoiesis by down regulating GATA -3 and upregulating erythropoietic-related genes such as GATA-1, Flk-1 and Epo-R. The morphological characteristics of cells after secondary differentiation showed enhanced production of erythrocytic precursors in dexamethasone containing media, which corresponded with molecular studies. Further studies will address the role of wnt/β-catenin and E-cadherin in this process.
Gene Expression Analysis ofIn VitroCocultures to Study Interactions between Breast Epithelium and Stroma
