ALBERT

Description: Human induced pluripotent stem cells (iPSCs) bearing monogenic mutations have great potential for modeling disease phenotypes, screening candidate drugs, and cell replacement therapy provided the underlying disease-causing mutation can be corrected. Here, we report a homologous recombination-based approach to precisely correct the sickle cell disease (SCD) mutation in patient-derived iPSCs with 2 mutated β-globin alleles (βs/βs). Using a gene-targeting plasmid containing a loxP-flanked drug-resistant gene cassette to assist selection of rare targeted clones and zinc finger nucleases engineered to specifically stimulate homologous recombination at the βs locus, we achieved precise conversion of 1 mutated βs to the wild-type βA in SCD iPSCs. However, the resulting co-integration of the selection gene cassette into the first intron suppressed the corrected allele transcription. After Cre recombinase-mediated excision of this loxP-flanked selection gene cassette, we obtained “secondary” gene-corrected βs/βA heterozygous iPSCs that express at 25% to 40% level of the wild-type transcript when differentiated into erythrocytes. These data demonstrate that single nucleotide substitution in the human genome is feasible using human iPSCs. This study also provides a new strategy for gene therapy of monogenic diseases using patient-specific iPSCs, even if the underlying disease-causing mutation is not expressed in iPSCs.

Description: Abstract 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.

Description: Human induced pluripotent stem (iPS) cells derived from somatic cells hold promise to develop novel patient-specific cell therapies and research models for inherited and acquired diseases. We and others previously reprogrammed human adherent cells, such as postnatal fibroblasts to iPS cells, which resemble adherent embryonic stem cells. Here we report derivation of iPS cells from postnatal human blood cells and the potential of these pluripotent cells for disease modeling. Multiple human iPS cell lines were generated from previously frozen cord blood or adult CD34+ cells of healthy donors, and could be redirected to hematopoietic differentiation. Multiple iPS cell lines were also generated from peripheral blood CD34+ cells of 2 patients with myeloproliferative disorders (MPDs) who acquired the JAK2-V617F somatic mutation in their blood cells. The MPD-derived iPS cells containing the mutation appeared normal in phenotypes, karyotype, and pluripotency. After directed hematopoietic differentiation, the MPD-iPS cell-derived hematopoietic progenitor (CD34+CD45+) cells showed the increased erythropoiesis and gene expression of specific genes, recapitulating features of the primary CD34+ cells of the corresponding patient from whom the iPS cells were derived. These iPS cells provide a renewable cell source and a prospective hematopoiesis model for investigating MPD pathogenesis.

Description: Abstract 1589 Human induced pluripotent stem cells (iPSCs) that are functionally similar to embryonic stem cells (ESCs) hold great potential for cell and gene therapies, disease modeling and drug development. The earliest success was achieved by using adherent fibroblastic cells and retroviral vectors that transduce fibroblasts very efficiently. It is also highly desirable to reprogram postnatal blood cells, including those from cord blood (CB) and adult peripheral blood (PB), which are easily accessible and less exposed to environmental mutagens. In 2009, we and others have achieved the reprogramming of human postnatal blood cells using the 4 Yamanaka factors delivered by retroviral vectors. We also found that reprogramming efficiencies of CB and PB CD34+ cells are higher than age-matched fibroblasts or MSCs. This may result from an epigenetic profile of hematopoietic CD34+ cells that appears closer to iPSCs/ESCs than that of fibroblasts/MSCs to iPSCs/ESCs. To generate integration-free iPSCs that produce hematopoietic progeny efficiently, we attempted to reprogram adult PB as well as CB cells by OriP/EBNA1 episomal vectors, which were used previously to reprogram foreskin fibroblasts albeit at a low efficiency (Yu/Thomson, 2009). When one of the best combinations (#6, 3 plasmids) was used, 1–3 candidate iPSC clones per 1 million cells were obtained as reported (Yu/Thomson, 2009). We and others found that the efficiency of generating iPS clones was even lower with human adult somatic cells by the 3 vectors. To improve, we constructed a new episomal reprogramming vector system using 1–2 OriP/EBNA1 plasmids. One (pEB-C5) expresses 5 factors (OCT4/SOX2/KLF4/Myc/LIN28), and the second expresses SV40 T antigen (Tg). CB and adult PB CD34+ cells were first cultured for 4 days and expanded ≥4-folds. The expanded cells (1 million) were then transfected once by the 1–2 new OriP/EBNA1 plasmids we constructed. Fourteen days later, we obtained on average 250 and 9 TRA-1-60+, iPSC-like colonies from CB and adult PB cells, respectively, when both pCB-C5 and pEB-Tg were used. A single plasmid (pEB-C5) can also generate iPSCs although the efficiency is ∼4-folds lower. Five characterized iPSC lines derived from CB and adult PB CD34+ cells (with or without Tg) are karyotypically normal and pluripotent. After successful reprogramming and expansion, episomal DNA is gradually lost in proliferating iPSCs. After serial expansions for 11–12 passages, vector DNA was undetectable either as episomes or in the genome of the 5 iPSC lines. We next extended this approach to reprogram un-fractionated adult PB mononuclear cells (PBMCs) including those from a sickle cell patient (SCDB003). To achieve better cell proliferation that is critical to iPSC production, we used a culture condition that favors the formation and proliferation of erythroblasts from PBMCs. PBMCs purified by standard Ficoll gradient were cultured in a serum-free condition with cytokines SCF, EPO and IL-3. Although cell death was observed and cell number decreased significantly in the first 4 days, equal or more cells than input were obtained by day 8. The expanded cells morphologically resemble pro-erythroblast cells, and express high-level CD71. Less than 1.5% of them express markers of T cells (CD3, CD2, CD4 and CD8) and B cells (CD19 and CD20). When 2×106 expanded SCDB003 cells (achievable from PBMCs in 1 ml or less PB) were transfected by the 2 OriP/EBNA1 plasmids and reprogrammed in the presence of butyrate, we observed 8 colonies at day 14 that are TRA-1-60+ and iPSC-like. The second plasmid (pEB-Tg) was not essential although it enhanced the efficiency by ∼4 folds. We picked and characterized 3 iPSC-like colonies derived from PBMCs with or without Tg. All of them express pluripotency markers and behave as typical iPSCs. So far we do not have evidence if they are derived from committed T or B cells that somatic mutations altered and rearranged their genomes. We are currently examining karyotypes, in vivo pluripotency, and status of episomal vectors in 3 PBMC-derived iPSCs. As compared to recent studies using viruses that preferentially reprogram human T cells with a rearranged genome, our method of using 1–2 plasmids is virus-free and genomic alteration-free. The ability to obtain integration-free human iPSCs from a few ml PB by 1–2 plasmids will greatly accelerate uses of iPSCs in both research and future clinical applications, epically for blood disease modeling and treatment. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 975 Every second, a healthy human body produces ∼2 million red blood cells (RBCs), an impossible feat for patients suffering from certain anemias not alleviated by erythropoietin (EPO) therapy. Instead, they rely on blood transfusions. Currently, all blood supplies are from donors, with inherent infection risks and supply uncertainty. Furthermore, some patients (such as those with sickle cell anemia) need frequent transfusion of RBC concentrates from best-matched donors, which are difficult to find. The production of cultured human RBCs in the quantities required for transfusion therapy (about 2 trillion RBCs in one transfusion unit of blood) will have great potential for improving healthcare worldwide. Large-scale production of cultured RBCs from isolated human CD34+ post-natal hematopoietic stem/progenitor cells (HSPCs) has achieved some success in the past decade. However, many challenges remain, such as determining the best method to enhance the cell number expansion and improve the efficiency of terminal maturation. CD34+ mononuclear cells (MNCs) contain HSPCs that have high proliferative capacity, but are very rare (

Description: Abstract 812 Human induced pluripotent stem (iPS) cells derived from somatic cells hold promise to develop novel patient-specific cell therapies and research models for inherited and acquired diseases. We and others previously reprogrammed human adherent cells such as postnatal fibroblasts to iPS cells that resemble adherent human embryonic stem (hES) cells. It is also highly desirable to reprogram blood cells that are easily accessible and less exposed to environmental mutagens. The large numbers of umbilical cord blood (CB) cells that are collected and stored in multiple cell banks are examples that could be used as a source of either autologous or allogeneic but histo-compatible iPS cell lines. As in vitro expansion of hematopoietic stem/progenitor cells from CB and adult sources remains a challenge, unlimited expansion of derived iPS cells in combination with further optimized hematopoietic differentiation methods should provide a vital alternative to amplify histo-compatible blood stem cells for blood/BM transplantation purposes. More critically, the ability to reprogram blood cells is essential if one wishes to generate iPS cells containing somatic mutations that are restricted to the blood cells and found in acquired hematological disorders, such as myeloproliferative disorders (MPDs), in order to investigate their pathogenesis. The BCR/ABL-negative MPDs, which include polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF), are a heterogeneous group of diseases characterized by increased proliferation of erythroid, megakaryocytic and myeloid lineages alone or in combination. The acquired common somatic mutation JAK2-V617F is present in 〉95% of PV, and ∼50% of ET and PMF patients. The discovery of this mutation in 2005 has significantly improved our understanding of MPD mechanisms and has intensified the search for drug(s) that may effectively inhibit this aberrant kinase activation. However, new disease models are needed to answer questions it raised such as how gene dosages of JAK2-V617F and other pre-disposing mutations affect the MPD pathogenesis. Here we report the derivation of iPS cells from postnatal human blood cells and the potential of these pluripotent cells for hematopoietic differentiation and disease modeling. Multiple human iPS cell lines were generated from previously frozen cord blood, adult peripheral blood and marrow CD34+ cells of healthy donors. The hematopoietic differentiation potential of these human iPS cells was examined by an improved method of EB formation and differentiation under a feeder- and serum-free condition. After two weeks of treatment, the cells were harvested and analyzed by both hematopoietic colony-forming assays and FACS for the presence of hematopoietic markers. Both myeloid and erythroid colonies were detected as we have observed using hES cells. By FACS analysis, CD45+ (27%-64%) and CD43+ (36%-60%) hematopoietic cells co-expressing undetectable to intermediate levels of CD34 marker were also observed. Multiple iPS cell lines were also generated from peripheral blood CD34+ cells of two patients with myeloproliferative disorders (MPDs) who acquired the JAK2-V617F somatic mutation in their blood cells. The MPD-derived iPS cells containing the mutation appeared normal in phenotypes, karyotype and pluripotency. To determine if these MPD iPS cell lines could be used as a model to study abnormal human hematopoiesis, we used the same serum-free differentiation protocol to direct them into hematopoietic lineages. Similar to the increased erythropoiesis of hematopoietic progenitor (CD34+) cells isolated from PV patients, including one subject whose blood-derived iPS cells were used in this study, re-differentiated hematopoietic progenitor (CD34+CD45+) cells from the PV-iPS cells showed enhanced erythropoiesis as compared to those from the iPS cells derived from normal CD34+ cells. They also showed a gene expression pattern similar to the primary CD34+ cells from the PV patient. These iPS cells thus provide a renewable cell source and a prospective model for investigating MPD pathogenesis. In combination with the gene targeting technology we recently described, human iPS cell lines derived from patients and subsequent hematopoietic differentiation technologies provide a novel model for investigations of various blood diseases with either acquired or inherited mutations. Disclosures: No relevant conflicts of interest to declare.

Description: We have developed induced pluripotent stem cells (iPSCs) from a patient with X-linked chronic granulomatous disease (X-CGD), a defect of neutrophil microbicidal reactive oxygen species (ROS) generation resulting from gp91phox deficiency. We demonstrated that mature neutrophils differentiated from X-CGD iPSCs lack ROS production, reproducing the pathognomonic CGD cellular phenotype. Targeted gene transfer into iPSCs, with subsequent selection and full characterization to ensure no off-target changes, holds promise for correction of monogenic diseases without the insertional mutagenesis caused by multisite integration of viral or plasmid vectors. Zinc finger nuclease–mediated gene targeting of a single-copy gp91phox therapeutic minigene into one allele of the “safe harbor” AAVS1 locus in X-CGD iPSCs without off-target inserts resulted in sustained expression of gp91phox and substantially restored neutrophil ROS production. Our findings demonstrate how precise gene targeting may be applied to correction of X-CGD using zinc finger nuclease and patient iPSCs.