Generation of GPI Anchor Deficient Blood Cells From Human iPSCs.

The PIG-A gene is an X-lined gene required for biosynthesis of glycosylphosphatidylinositol anchor proteins (GPI-AP). The PIG-A mutations can be acquired or inherited. Acquired mutations in hematopoietic stem cells leads to paroxysmal nocturnal hemoglobinuria (PNH). Germ line null mutations are embryonic lethal but hypomorphic mutations cause severe developmental abnormalities, intractable seizures and early death. To better understand the consequence of PIG-A mutations in human development and hematopoiesis, we established human induced pluripotent stem cell lines (hiPSC) that lack PIG-A expression and are therefore GPI-AP deficient. We established an inducible system for conditional expression of the PIG-A gene within PIG-A null hiPSCs. This system allowed us to generate GPI anchor deficient (GPI-AP^-) blood cells in multiple lineages.

We used a gene-targeting technology to knock out the PIG-A gene in male (XY) hiPSC derived from human somatic cells. F5HR (PIG-A^null, GPI-AP⁻) cells were transduced with pLV-PIG-A lentiviral vector (LV) containing the full-length human PIG-A transgene. F5HR (PIG-A^null, GPI-AP⁻) cells were co-transduced by two LV vectors: the regulator of pLV-tTR-KRAB (tTS/RFP) containing the tetracycline (tet)-SUPER inducible transgene expression system and the other of pLV-PIG-A expressing human PIG-A cDNA controlled by the human EF1α promoter. Once Dox is added, PIG-A gene expression is turned on. This inducible cell line (F5HKP) allows us to explore the function of the PIG-A gene during embryoid body (EB) generation and blood cell differentiation.

We examined the potential of F5 cells and F5HR cells (PIG-A^null) to form EBs and generate hematopoietic colonies. F5 hiPSC, but not F5HR hiPSC, cultured in serum free medium with BMP-4, VEGF and SCF formed morphologically normal EBs with evidence of blood-like cells surrounding the EB after 12–15 days in culture. Transduction of the F5HR cells with PIG-A restored the ability of the F5HR cells to form normal EBs and produce blood-like cells. Cells derived from the day 15 EB were further analyzed for the expression of the hematopoietic cell surface proteins (CD34 and CD45) and for the ability to generate hematopoietic colonies. The PIG-A mutant F5HR cells did not express CD59, CD34, or CD45 and were unable to generate hematopoietic colonies. We next induced mesodermal differentiation in the F5 and F5HR hiPSC and assessed cell surface expression of CD56, KDR and CD34 on days 0, 3, 7, and 10 during mesoderm differentiation. Surface expression of CD56 and KDR was virtually absent from the day 3 EBs derived from the F5HR iPS cells. CD34 expression by day 10 of mesoderm differentiation was negligible. Restoration of the PIG-A gene into F5HR cells restored the normal expression pattern of all three early mesodermal markers. Next, we transduced the F5HR iPS cells with our inducible PIG-A expression system to establish the F5HKP hiPSC. CD59⁺ undifferentiated F5HKP cells were cultured in mesoderm inducing medium with or without Dox for up to 14 days and the percentage of EB with blood-like cells in 96-well plates were counted. F5HKP hiPSC cultured without Dox lost GPI-AP expression, formed abnormal EBs, and did not generate blood-like cells. However, in presence of Dox for 14 days, F5HKP cells remained GPI-AP⁺ and made morphologically normal EB with blood-like cells. When Dox was removed from the culture conditions after 14 days myeloid and erythroid lineages were shown to be GPI-AP^neg. GPI-AP^negblood cells derived from F5HKP cells were show to be resistant to proaerolysin, a toxin that uses GPI-APs as its receptor.

Using PIG-A gene targeting in hiPSCs and an inducible PIG-A expression system, we have established a conditional PIG-A knockout model that allows for the production of GPI-AP^neg human blood cells. These studies show that PIG-A null hiPSCs are unable to form blood. The initial block is at the generation of mesodermal progenitors that express CD56, predating the deficiency of precursor cells expressing KDR and CD34. However, transient expression of GPI-APs during hematopoietic differentiation allowed us to generate GPI-AP^neg blood cells in multiple hematopoietic lineages. This conditional PIG-A knockout system should be a valuable tool for disease modeling PNH and other congenital diseases associated with GPI-AP deficiency.

No relevant conflicts of interest to declare.