MURINE EMBRYONIC STEM-CELLS WITHOUT PIG-A GENE ACTIVITY ARE COMPETENTFOR HEMATOPOIESIS WITH THE PNH PHENOTYPE BUT NOT FOR CLONAL EXPANSION

Paroxysmal nocturnal hemoglobinuria (PNH) develops in patients who hav e had a somatic mutation in the X-linked PIG-A gene in a hematopoietic stem cell; as a result, a proportion of blood cells are deficient in all glycosyl phosphatidylinositol (GPI)-anchored proteins, Although th e PIG-A mutation explains the phenotype of PNH cells, the mechanism en abling the PNH stem cell to expand is not clear, To examine this growt h behavior, and to investigate the role of GPI-linked proteins in hema topoietic differentiation, we have inactivated the pig-a gene by homol ogous recombination in mouse embryonic stem (ES) cells, In mouse chime ras, pig-a- ES cells were able to contribute to hematopoiesis and to d ifferentiate into mature red cells, granulocytes, and lymphocytes with the PNH phenotype, The proportion of PNH red cells was substantial in the fetus, but decreased rapidly after birth, Likewise, PNH granulocy tes could only be demonstrated in the young mouse. In contrast, the pe rcentage of lymphocytes deficient in GPI-linked proteins was more stab le, In vitro, pig-a- ES cells were able to form pig-a- embryoid bodies and to undergo hematopoietic (erythroid and myeloid) differentiation. The number and the percentage of pig-a- embryoid bodies with hematopo ietic differentiation, however, were significantly lower when compared with wild-type embryoid bodies. Our findings demonstrate that murine ES cells with a nonfunctional pig-a gene are competent for hematopoies is, and give rise to blood cells with the PNH phenotype, pig-a inactiv ation on its own, however, does not confer a proliferative advantage t o the hematopoietic stem cell. This provides direct evidence for the n otion that some additional factor(s) are needed for the expansion of t he mutant clone in patients with PNH.