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J Clin Invest. 1997 September 1; 100(5): 1028–1036.
PMCID: PMC508277

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


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- 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- 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 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.

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Selected References

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  • Dacie JV, Lewis SM. Paroxysmal nocturnal haemoglobinuria: clinical manifestations, haematology, and nature of the disease. Ser Haematol. 1972;5(3):3–23. [PubMed]
  • Takeda J, Miyata T, Kawagoe K, Iida Y, Endo Y, Fujita T, Takahashi M, Kitani T, Kinoshita T. Deficiency of the GPI anchor caused by a somatic mutation of the PIG-A gene in paroxysmal nocturnal hemoglobinuria. Cell. 1993 May 21;73(4):703–711. [PubMed]
  • Bessler M, Mason PJ, Hillmen P, Miyata T, Yamada N, Takeda J, Luzzatto L, Kinoshita T. Paroxysmal nocturnal haemoglobinuria (PNH) is caused by somatic mutations in the PIG-A gene. EMBO J. 1994 Jan 1;13(1):110–117. [PubMed]
  • Rosse WF. Phosphatidylinositol-linked proteins and paroxysmal nocturnal hemoglobinuria. Blood. 1990 Apr 15;75(8):1595–1601. [PubMed]
  • Kinoshita T, Inoue N, Takeda J. Defective glycosyl phosphatidylinositol anchor synthesis and paroxysmal nocturnal hemoglobinuria. Adv Immunol. 1995;60:57–103. [PubMed]
  • Masterson WJ, Doering TL, Hart GW, Englund PT. A novel pathway for glycan assembly: biosynthesis of the glycosyl-phosphatidylinositol anchor of the trypanosome variant surface glycoprotein. Cell. 1989 Mar 10;56(5):793–800. [PubMed]
  • Bessler M, Hillmen P, Longo L, Luzzatto L, Mason PJ. Genomic organization of the X-linked gene (PIG-A) that is mutated in paroxysmal nocturnal haemoglobinuria and of a related autosomal pseudogene mapped to 12q21. Hum Mol Genet. 1994 May;3(5):751–757. [PubMed]
  • Watanabe R, Kinoshita T, Masaki R, Yamamoto A, Takeda J, Inoue N. PIG-A and PIG-H, which participate in glycosylphosphatidylinositol anchor biosynthesis, form a protein complex in the endoplasmic reticulum. J Biol Chem. 1996 Oct 25;271(43):26868–26875. [PubMed]
  • Luzzatto L, Bessler M. The dual pathogenesis of paroxysmal nocturnal hemoglobinuria. Curr Opin Hematol. 1996 Mar;3(2):101–110. [PubMed]
  • Rosse WF, Ware RE. The molecular basis of paroxysmal nocturnal hemoglobinuria. Blood. 1995 Nov 1;86(9):3277–3286. [PubMed]
  • Rosse WF, Dacie JV. Immune lysis of normal human and paroxysmal nocturnal hemoglobinuria (PNH) red blood cells. I. The sensitivity of PNH red cells to lysis by complement and specific antibody. J Clin Invest. 1966 May;45(5):736–748. [PMC free article] [PubMed]
  • van Kamp H, Landegent JE, Jansen RP, Willemze R, Fibbe WE. Clonal hematopoiesis in patients with acquired aplastic anemia. Blood. 1991 Dec 15;78(12):3209–3214. [PubMed]
  • Dameshek W. Riddle: what do aplastic anemia, paroxysmal nocturnal hemoglobinuria (PNH) and "hypoplastic" leukemia have in common? Blood. 1967 Aug;30(2):251–254. [PubMed]
  • Lewis SM, Dacie JV. The aplastic anaemia--paroxysmal nocturnal haemoglobinuria syndrome. Br J Haematol. 1967 Mar;13(2):236–251. [PubMed]
  • Rotoli B, Luzzatto L. Paroxysmal nocturnal haemoglobinuria. Baillieres Clin Haematol. 1989 Jan;2(1):113–138. [PubMed]
  • Luzzatto L, Bessler M, Rotoli B. Somatic mutations in paroxysmal nocturnal hemoglobinuria: a blessing in disguise? Cell. 1997 Jan 10;88(1):1–4. [PubMed]
  • Bessler M, Mason P, Hillmen P, Luzzatto L. Somatic mutations and cellular selection in paroxysmal nocturnal haemoglobinuria. Lancet. 1994 Apr 16;343(8903):951–953. [PubMed]
  • Swiatek PJ, Gridley T. Perinatal lethality and defects in hindbrain development in mice homozygous for a targeted mutation of the zinc finger gene Krox20. Genes Dev. 1993 Nov;7(11):2071–2084. [PubMed]
  • Kawagoe K, Takeda J, Endo Y, Kinoshita T. Molecular cloning of murine pig-a, a gene for GPI-anchor biosynthesis, and demonstration of interspecies conservation of its structure, function, and genetic locus. Genomics. 1994 Oct;23(3):566–574. [PubMed]
  • Laird PW, Zijderveld A, Linders K, Rudnicki MA, Jaenisch R, Berns A. Simplified mammalian DNA isolation procedure. Nucleic Acids Res. 1991 Aug 11;19(15):4293–4293. [PMC free article] [PubMed]
  • Pevny L, Lin CS, D'Agati V, Simon MC, Orkin SH, Costantini F. Development of hematopoietic cells lacking transcription factor GATA-1. Development. 1995 Jan;121(1):163–172. [PubMed]
  • Wiles MV, Keller G. Multiple hematopoietic lineages develop from embryonic stem (ES) cells in culture. Development. 1991 Feb;111(2):259–267. [PubMed]
  • Stowe HD, Wagner JL, Pick JR. A debilitating fatal murine dermatitis. Lab Anim Sci. 1971 Dec;21(6):892–897. [PubMed]
  • HogenEsch H, Gijbels MJ, Offerman E, van Hooft J, van Bekkum DW, Zurcher C. A spontaneous mutation characterized by chronic proliferative dermatitis in C57BL mice. Am J Pathol. 1993 Sep;143(3):972–982. [PubMed]
  • Wiles MV. Embryonic stem cell differentiation in vitro. Methods Enzymol. 1993;225:900–918. [PubMed]
  • Kawagoe K, Kitamura D, Okabe M, Taniuchi I, Ikawa M, Watanabe T, Kinoshita T, Takeda J. Glycosylphosphatidylinositol-anchor-deficient mice: implications for clonal dominance of mutant cells in paroxysmal nocturnal hemoglobinuria. Blood. 1996 May 1;87(9):3600–3606. [PubMed]
  • Medvinsky A, Dzierzak E. Definitive hematopoiesis is autonomously initiated by the AGM region. Cell. 1996 Sep 20;86(6):897–906. [PubMed]
  • Moore MA, Metcalf D. Ontogeny of the haemopoietic system: yolk sac origin of in vivo and in vitro colony forming cells in the developing mouse embryo. Br J Haematol. 1970 Mar;18(3):279–296. [PubMed]
  • Gordon MY, Lewis JL, Marley SB, Grand FH, Goldman JM. Stromal cells negatively regulate primitive haemopoietic progenitor cell activation via a phosphatidylinositol-anchored cell adhesion/signalling mechanism. Br J Haematol. 1997 Mar;96(3):647–653. [PubMed]
  • Hillmen P, Lewis SM, Bessler M, Luzzatto L, Dacie JV. Natural history of paroxysmal nocturnal hemoglobinuria. N Engl J Med. 1995 Nov 9;333(19):1253–1258. [PubMed]
  • Doetschman TC, Eistetter H, Katz M, Schmidt W, Kemler R. The in vitro development of blastocyst-derived embryonic stem cell lines: formation of visceral yolk sac, blood islands and myocardium. J Embryol Exp Morphol. 1985 Jun;87:27–45. [PubMed]
  • Gordon MY, Riley GP, Watt SM, Greaves MF. Compartmentalization of a haematopoietic growth factor (GM-CSF) by glycosaminoglycans in the bone marrow microenvironment. Nature. 326(6111):403–405. [PubMed]
  • Roberts R, Gallagher J, Spooncer E, Allen TD, Bloomfield F, Dexter TM. Heparan sulphate bound growth factors: a mechanism for stromal cell mediated haemopoiesis. Nature. 1988 Mar 24;332(6162):376–378. [PubMed]
  • Brunner G, Gabrilove J, Rifkin DB, Wilson EL. Phospholipase C release of basic fibroblast growth factor from human bone marrow cultures as a biologically active complex with a phosphatidylinositol-anchored heparan sulfate proteoglycan. J Cell Biol. 1991 Sep;114(6):1275–1283. [PMC free article] [PubMed]
  • Dunn DE, Yu J, Nagarajan S, Devetten M, Weichold FF, Medof ME, Young NS, Liu JM. A knock-out model of paroxysmal nocturnal hemoglobinuria: Pig-a(-) hematopoiesis is reconstituted following intercellular transfer of GPI-anchored proteins. Proc Natl Acad Sci U S A. 1996 Jul 23;93(15):7938–7943. [PubMed]
  • Bielinska M, Narita N, Heikinheimo M, Porter SB, Wilson DB. Erythropoiesis and vasculogenesis in embryoid bodies lacking visceral yolk sac endoderm. Blood. 1996 Nov 15;88(10):3720–3730. [PubMed]

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