Phosphatidylinositol glycan class A (PIGA) is involved in the first step of glycosylphosphatidylinositol (GPI) biosynthesis. Many proteins, including CD55 and CD59, are anchored to the cell by GPI. Loss of CD55 and CD59 on erythrocytes causes complement-mediated lysis in paroxysmal nocturnal hemoglobinuria (PNH), a disease that manifests after clonal expansion of hematopoietic cells with somatic PIGA mutations. Although somatic PIGA mutations have been identified in many PNH patients, it has been proposed that germline mutations are lethal. We report a family with an X-linked lethal disorder involving cleft palate, neonatal seizures, contractures, central nervous system (CNS) structural malformations, and other anomalies. An X chromosome exome next-generation sequencing screen identified a single nonsense PIGA mutation, c.1234C>T, which predicts p.Arg412(∗). This variant segregated with disease and carrier status in the family, is similar to mutations known to cause PNH as a result of PIGA dysfunction, and was absent in 409 controls. PIGA-null mutations are thought to be embryonic lethal, suggesting that p.Arg412(∗) PIGA has residual function. Transfection of a mutant p.Arg412(∗) PIGA construct into PIGA-null cells showed partial restoration of GPI-anchored proteins. The genetic data show that the c.1234C>T (p.Arg412(∗)) mutation is present in an affected child, is linked to the affected chromosome in this family, is rare in the population, and results in reduced, but not absent, biosynthesis of GPI anchors. We conclude that c.1234C>T in PIGA results in the lethal X-linked phenotype recognized in the reported family.

Disease-specific induced pluripotent stem cells (iPSCs) provide an unprecedented opportunity to establish novel disease models and accelerate drug development using distinct tissue target cells generated from isogenic iPSC lines with and without disease-causing mutations. To realize the potential of iPSCs in modeling acquired diseases which are usually heterogeneous, we have generated multiple iPSC lines including two lines that are JAK2-wild-type and four lines homozygous for JAK2-V617F somatic mutation from a single polycythemia vera (PV) patient blood. In vitro differentiation of the same patient-derived iPSC lines have demonstrated the differential contributions of their parental hematopoietic clones to the abnormal erythropoiesis including the formation of endogenous erythroid colonies. This iPSC approach thus may provide unique and valuable insights into the genetic events responsible for disease development. To examine the potential of iPSCs in drug testing, we generated isogenic hematopoietic progenitors and erythroblasts from the same iPSC lines derived from PV patients and normal donors. Their response to three clinical JAK inhibitors, INCB018424 (Ruxolitinib), TG101348 (SAR302503), and the more recent CYT387 was evaluated. All three drugs similarly inhibited erythropoiesis from normal and PV iPSC lines containing the wild-type JAK2 genotype, as well as those containing a homozygous or heterozygous JAK2-V617F activating mutation that showed increased erythropoiesis without a JAK inhibitor. However, the JAK inhibitors had less inhibitory effect on the self-renewal of CD341 hematopoietic progenitors. The iPSC-mediated disease modeling thus underlies the ineffectiveness of the current JAK inhibitors and provides a modeling system to develop better targeted therapies for the JAK2 mutated hematopoiesis.

Primordial germ cells (PGCs) share many properties with embryonic stem cells (ESCs) and innately express several key pluripotency-controlling factors, including OCT4, NANOG, and LIN28. Therefore, PGCs may provide a simple and efficient model for studying somatic cell reprogramming to induced pluripotent stem cells (iPSCs), especially in determining the regulatory mechanisms that fundamentally define pluripotency. Here, we report a novel model of PGC reprogramming to generate iPSCs via transfection with SOX2 and OCT4 using integrative lentiviral. We also show the feasibility of using nonintegrative approaches for generating iPSC from PGCs using only these two factors. We show that human PGCs express endogenous levels of KLF4 and C-MYC protein at levels similar to embryonic germ cells (EGCs) but lower levels of SOX2 and OCT4. Transfection with both SOX2 and OCT4 together was required to induce PGCs to a pluripotent state at an efficiency of 1.71%, and the further addition of C-MYC increased the efficiency to 2.33%. Immunohistochemical analyses of the SOderived PGC-iPSCs revealed that these cells were more similar to ESCs than EGCs regarding both colony morphology and molecular characterization. Although leukemia inhibitory factor (LIF) was not required for the generation of PGC-iPSCs like EGCs, the presence of LIF combined with ectopic exposure to C-MYC yielded higher efficiencies. Additionally, the SO-derived PGC-iPSCs exhibited differentiation into representative cell types from all three germ layers in vitro and successfully formed teratomas in vivo. Several lines were generated that were karyotypically stable for up to 24 subcultures. Their derivation efficiency and survival in culture significantly supersedes that of EGCs, demonstrating their utility as a powerful model for studying factors regulating pluripotency in future studies.

Derivation of pluripotent stem cells (iPSCs) induced from somatic cell types and the subsequent genetic modifications of disease-specific or patient-specific iPSCs are crucial steps in their applications for disease modeling as well as future cell and gene therapies. Conventional procedures of these processes require co-culture with primary mouse embryonic fibroblasts (MEFs) to support self-renewal and clonal growth of human iPSCs as well as embryonic stem cells (ESCs). However, the variability of MEF quality affects the efficiencies of all these steps. Furthermore, animal sourced feeders may hinder the clinical applications of human stem cells. In order to overcome these hurdles, we established immortalized human feeder cell lines by stably expressing human telomerase reverse transcriptase, Wnt3a, and drug resistance genes in adult mesenchymal stem cells. Here, we show that these immortalized human feeders support efficient derivation of virus-free, integration-free human iPSCs and long-term expansion of human iPSCs and ESCs. Moreover, the drug-resistance feature of these feeders also supports nonviral gene transfer and expression at a high efficiency, mediated by piggyBac DNA transposition. Importantly, these human feeders exhibit superior ability over MEFs in supporting homologous recombination-mediated gene targeting in human iPSCs, allowing us to efficiently target a transgene into the AAVS1 safe harbor locus in recently derived integration-free iPSCs. Our results have great implications in disease modeling and translational applications of human iPSCs, as these engineered human cell lines provide a more efficient tool for genetic modifications and a safer alternative for supporting self-renewal of human iPSCs and ESCs.

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2358 Background: 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. Method: We used a gene-targeting technology to knock out the PIG-A gene in male (XY) hiPSC derived from human somatic cells. F5HR (PIG-Anull, GPI-AP−) cells were transduced with pLV-PIG-A lentiviral vector (LV) containing the full-length human PIG-A transgene. F5HR (PIG-Anull, 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. Results: We examined the potential of F5 cells and F5HR cells (PIG-Anull) 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-APneg. GPI-APnegblood cells derived from F5HKP cells were show to be resistant to proaerolysin, a toxin that uses GPI-APs as its receptor. Conclusion: 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-APneg 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-APneg 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. Disclosures: No relevant conflicts of interest to declare.

2826 Polycythemia vera (PV) is a clonal blood disorder arising from a multipotent hematopoietic stem cell (HSC) associated in ∼95% of patients with the acquired somatic mutation, JAK2 V617F. Although important for the PV phenotype, we and others demonstrated that the JAK2 V617F mutation is not the initial and causative somatic event in PV pathogenesis. One of the major challenges of studying the molecular events in PV is to isolate and expand the disease-initiating HSC clones in vitro. To overcome this hurdle, we have utilized the recently developed induced pluripotent stem cell (iPSC) technology to generate disease-specific iPSC lines that preserve the genetic identities of patient HSC clones. We previously demonstrated that interferon (IFN) a is the only therapy that converts PV hematopoiesis from clonal to polyclonal (Liu, Blood 2003). A female patient with typical PV and a high allele burden (99%) of JAK2 V617F, and ∼1% of wild-type JAK2 was treated with peg-IFNa. JAK2 allele burden decreased to ∼65%, yet the majority of her myeloid cells remained clonal. Using her blood and bone marrow progenitors as well as blood samples from other PV patients, we generated dozens of iPSC clones by retroviral or episomal vectors with several distinct JAK2 genotypes (see Table below). We examined the erythroid differentiation of 6 representative PV-iPSC lines and normal control iPSCs. The hematopoietic progenitor cells (HPCs) derived from JAK2 V617F iPSCs had enhanced erythropoiesis compared to wild-type JAK2 iPSC cells. Additionally, some EPO-independent BFU-Es also formed from homozygous JAK2 V617F iPSCs, the hallmark of PV erythropoiesis. Using a quantitative X-chromosome transcriptional assay (Swierczek, Blood 2008), we examined the clonality of the iPSC clones (with and without the JAK2 mutation) derived from this single female patient and showed the same single X-chromosome usage in all clones as in her native PV granulocytes and platelets. These data indicate that epigenetic X-chromosome silencing is not reverted in the process of generating iPSC clones. Whether these two JAK2 V617F-negative iPSC lines originated from the same PV clone or from dormant normal HSC cells cannot be yet discerned but their EPO sensitivity is currently under analysis. Analyses of whole exome sequencing of these iPS clones as well as their germ-line control are currently underway. Additionally, whole genome and epigenome analyses and high density expression array of these iPSC clones would further characterize the clonal evolution of PV. These data underscore the heterogeneity of somatic mutations within single PV patient. These studies will lead to a better understanding of the genetic lesions in PV-initiating clones. (Note: The last two authors are both considered senior authors for this work)Table.Human iPSC lines from a female PV patient and healthy donorsRepresentative iPS clone (# of clones characterized)DonorParental cell typeJAK2 WT alleleJAK2 V617F alleleKaryotypePVB1.4 (3)PVPB MNC0246, XXPVB1.1 (4)PB MNC1247, XX, +der(1;9)(q10;p10)PVB1.11 (2)PB MNC2046, XXPVM1.1 (2)BM MSC2046, XXBC1 (>5)Healthy donorBM CD34+2046, XYPB: peripheral blood; BM: bone marrow; MSC: mesenchymal stem cell. Disclosures: No relevant conflicts of interest to declare.