Developmental Potentials of Human Embryonic Stem Cells Lacking PIG-A and GPI-Anchored Proteins.

Paroxysmal Nocturnal Hemoglobinuria (PNH) is a clonal disorder of hematopoietic stem cells (HSCs) acquiring mutations in the PIG-A gene. Its product (PIG-A protein) is required for biosynthesis of dozens of cell surface proteins to be linked to glycosyl-phosphatidyl-inositol (GPI) molecule and anchored on plasma membrane. The underlying mechanisms of PIG-A mutant clonal dominance in PNH patients and the close relationship of PNH to other bone marrow failure diseases (aplastic anemia and myelodysplasia syndrome) and leukemia remain unknown. Establishing a mutation of PIG-A gene in human HSCs from healthy donors remains unfeasible due to the current inability to expand and select clonal HSCs in culture. Although mouse models have been generated by disrupting conditionally the mouse Pig-a gene, the existing Pig-a null mice indeed lacking GPI-APs in blood cells did not replicate faithfully PNH pathological symptoms. To create a human cell-based, prospective experimental system to investigate the effects of PIG-A/GPI-AP deficiency and pathophysiology of PNH, we made mutated hES cells lacking GPI-APs. FACS analysis revealed that two independent hES clones lack cell-surface expression of CD55 and CD59, as well as CD90/Thy and Cripto that are preferentially expressed in undifferentiated hES cells. However, the cell-surface expression of these GPI-APs can be restored by a lentiviral vector inducibly expressing the PIG-A cDNA. Like mouse ES cells, lack of PIG-A/GPI-APs did not affect the growth of undifferentiated hES cells in culture. Unlike mouse ES cells, however, PIG-A/GPI-AP deficient hES cells formed embryoid bodies normally in culture. RT-PCR analysis of marker gene expression indicated that commitment to the 3 (somatic) germ layers appeared normal within embryonic bodies from either the mutated or wildtype hES cells. However, formation of extra-embryonic cells such as trophoblasts from the PIG-A/GPI-AP deficient hES cells is defective in both embryoid body formation and BMP4-induced assays. Expression of trophoectoderm-specific genes such as hCGalpha could not be induced in PIG-A/GPI-AP deficient hES cells upon BMP4 induced trophoectodermal differentiation. The induction of other trophoectoderm markers such as hCGbeta and CDX2 was also significantly reduced after the BMP4 treatment. The lack of tropho-ectoderm was further confirmed by lacking of hCG hormone production. The defect in trophoectoderm differentiation from the PIG-A/GPI-AP deficient hES cells was restored by the expression of the PIG-A cDNA in the mutated hES cells. For somatic cell differentiation, we are currently examining the effects of PIG-A/GPI-AP deficiency beyond the initial differentiation commitment during embryonic body formation stage. Along mesodermal and hematopoietic differentiation, we found that the PIG-A/GPI-AP deficiency in hES cells had little effect on the formation of CD34+ cells, a precursor cell population for human hematopoietic and endothelial lineages. We are currently examining the effects of PIG-A/GPI-AP deficiency on properties of hematopoietic cells derived from the mutated and normal hES cells. This study represents one of first cases that hES cells may provide a prospective research model to investigate genetic and developmental basis of human diseases.