ERYTHROID 5-AMINOLEVULINATE SYNTHASE IS REQUIRED FOR ERYTHROID-DIFFERENTIATION IN MOUSE EMBRYONIC STEM-CELLS

We have examined the induction of the enzymes of the heme biosynthetic pathway during erythroid differentiation of mouse embryonic stem (ES) cells, Following transfer to appropriate medium all of the pathway en zymes are induced within three days, Unlike differentiating mouse eryt hroleukemia cells (Lake-Bullock, H. and Dailey, H.A. Mol Cell Biol 13: 7122-7132, 1993), all of the enzymes appear to be induced simultaneous ly and not sequentially in differentiating ES cells, The role of eryth roid 5-aminolevulinate synthase (ALAS-2) in this differentiation proce ss was examined by disruption of the ALAS-2 gene, The targeting vector used for disruption replaced all of exons 4 to 6 with a selectable ne omycin resistance gene, The resulting genetically modified (ALAS-2 kno ckout) cells, as well as normal ES cells were used to study induction of heme biosynthesis. Following 10 days of culture in methylcellulose media significant morphological differences between the embryoid bodie s (EBs) of the two cell lines were observed. ES cells exhibited morpho logy of typical EBs with a dark field (blood island) in the center, wh ile ALAS-2 knockout ES cells developed very poorly both in size and sh ape, At 8 days of differentiation, only 3% of all EBs contained visibl e erythropoietic cells (i.e., stained positively for hemoglobin) in th e ALAS-2 knockout cell line, compared with 50% in ES cells. Most of th e genes in the heme synthetic pathway were expressed to a stable level within 3 to 6 days after induction in normal ES cells, while the ALAS -2 knockout cell line failed to significantly increase the level of ex pression of these genes, Fetal beta-globin mRNA was not detectable in the differentiating ALAS-2 knockout cells, whereas mRNA for this gene was detected in normal ES cells within 3 days of differentiation, Thes e results suggest that ALAS-2 is necessary for ES cell erythroid diffe rentiation and that there is an interrelationship between heme and glo bin synthesis in differentiating ES cells.