Reduced Hematopoietic Stem Cell Function in a Mouse Model with Conditional Pig-a Gene Deletion.

Abstract 2428 Poster Board II-405 Hematopoietic stem cells (HSCs) are capable of self-renewal as well as differentiation to produce progenitor cells of all hematopoietic lineages in order to sustain life long blood production. In normal inbred mice, HSCs survive multiple rounds of transplantation and extend function to over 100 months, 3-4 times the lifespan of a normal mouse (Harrison DE, Mech Ageing Dev, 1973). We assessed HSC functions in a murine model of paroxysmal nocturnal hemoglobinuria (PNH). PNH originates with a somatic mutation in a HSC of the X-linked phosphatydylinositol glycan class A (PIG-A) gene. We produced a mouse model with conditional Pig-a deletion (Pig-a -/- ) by cross-breeding mice carrying germline insertion of two lox sites flanking exon 6 of Pig-a gene with mice carrying the transgene Cre-recombinase driven by the human c-fes promoter. The resultant B6 Fes-cre-Pig-a flox (Pig-a -/- ) mice showed Pig-a gene inactivation specifically in hematopoietic cells. Mice survived without obvious abnormalities and did not show any sign of clinical PNH or bone marrow (BM) failure. In preliminary experiments, we confirmed that BM cells from Pig-a -/- donors had normal HSC functional ability to compete with BM cells from congenic B6-CD45.1 competitors in a competitive repopulation assay, as previously shown by another PNH mouse model with conditional Pig-a gene deletion (Keller P et al , J Exp Med, 2001). We then tested whether Pig-a deletion and the resultant GPI deficiency might affect HSC function long term after serial transplantation. Toward this end, we incubated BM cells from Pig-a -/- donors with aerolysin to lyse GPI-normal cells (GPI + ) and then transplanted the residual GPI-deficient (GPI - ) cells into lethally-irradiated normal C57BL/6 recipients. Within two months, GPI - BM cells engrafted efficiently in all recipients, and 95-100% of recipient T, B and granulocytes were GPI - cells by flow cytometry. After 11 months, we serially-transplanted the GPI - BM cells into lethally-irradiated secondary recipients. Phenotype analysis at one and three months after secondary transplantation also showed relative stable GPI - cell engraftment in T cells (29 ± 9% and 69 ± 29%), B cells (79 ± 6% and 72 ± 15%) and granulocytes (74 ± 11% and 70 ± 10%). After 8 months in the secondary recipients, we extracted BM cells from three secondary recipients with >70% GPI - cells in T, B, and granulocytes. Flow cytometry analysis showed that their BM cellularity was normal, but marrows contained three to five fold lower proportion of cells staining for the Kit + Sca1 + Lin - (KSL) markers. We pooled BM cells from these three secondary recipients and transplanted BM cells into lethally-irradiated third-round recipients. Three out of five recipients did not survive transplantation and died between one and two weeks after irradiation/transplantation. The remaining two recipients showed - cells at two months and - cells at seven months following transplantation, suggesting that functional ability of GPI - BM cells had been diminished or drastically reduced. In order to test the competitive repopulating capacity, we also used pooled BM cells from secondary recipients in a competitive repopulation assay, congenic B6-CD45.1 BM cells serving as competitors. There was essentially no engraftment from GPI - donor cells in all recipients, when measured at two and seven months following transplantation, indicating that, after 19 months and two rounds of transplantation, GPI - BM cells were unable to compete with control BM cells for engraftment. In conclusion, Pig-a deletion and lack of GPI-anchored proteins may cause reduced HSC function long-term. Thus, the expansion of the mutant HSC clone seen in PNH patients is unlikely related to any intrinsic self-renewal or proliferative advantage of the HSC clone carrying Pig-a mutation. Disclosures: No relevant conflicts of interest to declare.