A knock-out model of paroxysmal nocturnal hemoglobinuria: Pig-a(-) hematopoiesis is reconstituted following intercellular transfer of GPI-anchored proteins.
We created a "knockout" embryonic stem cell via targeted disruption of the phosphatidylinositol glycan class A (Pig-a) gene, resulting in loss of expression of cell surface glycosyl phosphatidylinositol-anchored proteins and reproducing the mutant phenotype of the human disease paroxysmal nocturnal hemoglobinuria. Morphogenesis of Pig-a- embryoid bodies (EB) in vitro was grossly aberrant and, unlike EB derived from normal embryonic stem cells, Pig-A EB produced no secondary hematopoietic colonies. Chimeric EB composed of control plus Pig-A- cells, however, appeared normal, and hematopoiesis from knock-out cells was reconstituted. Transfer in situ of glycosyl phosphatidylinositol-anchored proteins from normal to knock-out cells was demonstrated by two-color fluorescent analysis, suggesting a possible mechanism for these functional effects. Hematopoietic cells with mutated PIG-A genes in humans with paroxysmal nocturnal hemoglobinuria may be subject to comparable pathophysiologic processes and amenable to similar therapeutic protein transfer.
Dunn
D E
DE
Hematology Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892-1652, USA.
Yu
J
J
Nagarajan
S
S
Devetten
M
M
Weichold
F F
FF
Medof
M E
ME
Young
N S
NS
Liu
J M
JM
D000818:Animals; D002454:Cell Differentiation; D002478:Cells, Cultured; D018920:Coculture Techniques; D004622:Embryo, Mammalian; D017261:Glycosylphosphatidylinositols / Q000235:genetics* / Q000378:metabolism*; D006457:Hemoglobinuria, Paroxysmal / Q000235:genetics* / Q000473:pathology*; D006801:Humans; D008565:Membrane Proteins / Q000096:biosynthesis; D051379:Mice; D018345:Mice, Knockout; D008954:Models, Biological; D016254:Mutagenesis, Insertional; D013234:Stem Cells / Q000166:cytology*
D017261:Glycosylphosphatidylinositols; D008565:Membrane Proteins