Murine embryonic stem cells without pig-a gene activity are competent for hematopoiesis with the PNH phenotype but not for clonal expansion.

Abstract

Paroxysmal nocturnal hemoglobinuria (PNH) develops in patients who have 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 the PIG-A mutation explains the phenotype of PNH cells, the mechanism enabling the PNH stem cell to expand is not clear. To examine this growth behavior, and to investigate the role of GPI-linked proteins in hematopoietic differentiation, we have inactivated the pig-a gene by homologous recombination in mouse embryonic stem (ES) cells. In mouse chimeras, pig-a Ϫ ES cells were able to contribute to hematopoiesis and to differentiate 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 granulocytes could only be demonstrated in the young mouse. In contrast, the percentage of lymphocytes deficient in GPI-linked proteins was more stable. In vitro, pig-a Ϫ ES cells were able to form pig-a Ϫ embryoid bodies and to undergo hematopoietic (erythroid and myeloid) differentiation. The number and the percentage of pig-a Ϫ embryoid bodies with hematopoietic 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 hematopoiesis, and give rise to blood cells with the PNH phenotype. pig-a inactivation on its own, however, does not confer a proliferative advantage to the hematopoietic stem cell. This provides direct evidence for the notion that some additional factor(s) are needed for the expansion of the mutant clone in patients with PNH. ( J.

The cellular pathogenesis of paroxysmal nocturnal haemoglobinuria

“…The model has proved to have heuristic value, because we now know the molecular basis for the PNH abnormality, we know that PNH clones are present but do not expand in normal people, 11 and we know that PNH cells tend to disappear rather than to expand in mouse model systems. [14][15][16] Moreover, we have evidence that the conditional selective agent we had postulated may consist in T cell clones, because we have demonstrated such clones in a substantial proportion of PNH patients. 17 CD1d tetramer technology may help to prove that these clones have the properties that we have hypothetically ascribed to them.…”

“…13 (4) In mice with targeted inactivation of the pig-a gene the PNH cell population does not increase in time, and often tends to disappear. [14][15][16] This last finding does not support positive selection for mutant cells; by implication, it favours instead the notion that negative selection against non-mutant cells dominates the picture in human PNH patients.…”

The prothrombotic state in paroxysmal nocturnal hemoglobinuria: a multifaceted source

“…118,119 Generating PIG-A mutations has proven difficult, as PIG-A deletion in embryonic stem cells is lethal. 120 A PIG-A floxed mosaic mice model was generated with the coexistence of normal and mutated cells mimicking PNH patients, however, the PNH clone failed to clonally expand and produce PNH symptoms. 121 Kellet et al succeeded in creating a model with 100% of red blood cells being GPI-AP negative, however, no symptoms of PNH were observed.…”

The <i>in vivo</i> Pig-a gene mutation assay is applied to study the genotoxicity of procarbazine hydrochloride in Sprague-Dawley rats

“…Firstly, the Pig-a gene exhibits mono-allelic mutations that are detectable, and the mutant phenotype can be distinguished from wild-type cells by flow cytometry. Secondly, several lines of evidence indicate that no clear or strong selection for or against the two phenotypes exists in vivo (Araten et al, 2005;Rosti et al, 1997;Tremml et al, 1999;Ware et al, 1998;Yamamoto et al, 2002). Thirdly, the mutant and wild-type cells can circulate for at least one month in peripheral blood in rats (Miura et al, 2009).…”