Normal iPSC Research Articles

Synthetic Lethality CRISPR Screens in Isogenic iPSCs with Splicing Factor Gene Mutations

A major recent discovery from large-scale sequencing studies was that over half of Myelodysplastic Syndromes (MDS) patients harbor mutations in splicing factor (SF) genes. SF mutations are the most common class of mutations in MDS and occur early in the course of the disease. These strongly suggest that SF mutations are key to the pathogenesis of MDS and can provide new therapeutic opportunities. However, identifying the downstream effects of SF mutations that are critical for the development of MDS presents a big challenge due to the cellular and genetic heterogeneity of primary patient samples, the unavailability of immortalized cell lines harboring SF mutations in the native genomic context and the limited conservation of alternative splicing isoforms between mice and humans. We previously showed that SF-mutant induced pluripotent stem cells (iPSCs) generated from MDS patients recapitulate key features of the disease upon differentiation into hematopoietic lineages, including cellular phenotypes (increased cell death, decreased clonogenicity and dysplastic morphology), sensitivity to splicing modulating drugs and the altered RNA binding specificity of mutant SFs (Chang et al. Stem Cell Reports, 2018).To further investigate the effects of SF mutations, we used CRISPR to introduce each of the 3 main canonical SF mutations (SRSF2 P95L, SF3B1 K700E, U2AF1 S34F) in the same normal iPSC line N-2.12 that we previously derived and extensively characterized in terms of pluripotency, genetic integrity and hematopoietic differentiation potential. The derivative iPSC lines contain the 3 SF mutations in isogenic conditions in the context of a diploid genome, in a heterozygous state, with both the normal and mutant alleles expressed at physiological and equal levels. To uncover potential new therapeutic targets and gain insights into the downstream effects of SF mutations, we set up CRISPR knockout (KO) lethality screens in hematopoietic progenitor cells (HPCs) derived from these SF-mutant iPSCs. We began with a gRNA library containing 224 gRNAs targeting 57 kinase genes (4 gRNAs per gene). The library was assembled and packaged in a lentiviral backbone also expressing GFP. Cas9 together with mCherry was expressed from a separate lentiviral vector. iPSCs were differentiated along the hematopoietic lineage, transduced on day 11, coinciding with the onset of the emergence of CD34+/CD45+ HPCs, and further cultured for up to day 27 to allow “dropout” of lethal genes, read out by next-generation sequencing (NGS). We titrated the lentiviral vectors to obtain transduction efficiency of nearly 100% for the Cas9 vector and up to 40-50% for the gRNA library (in order to obtain the highest percentage of cells harboring a single gRNA) in 500,000 HPCs, to ensure representation of the library of at least 500 cells per gRNA. To avoid population bottlenecks, we ensured that at least 500,000 cells were present in the culture at all times. All library gRNAs were present in the transduced cells and their distribution correlated tightly with that of the lentiviral supernatant. Technical repeats of independently prepared DNA samples, independent PCR reactions and independently generated NGS reads showed high reproducibility and absence of batch effects.The library was screened in 3 independent clones harboring each of the 3 canonical SF mutations, as well as in 3 clones of the parental normal line. This design allows the identification of potential convergent genes or pathways downstream of the 3 SF mutations and exclusion of non-synthetically lethal targets (which would also drop out in the isogenic normal cells). CRISPR scores were calculated as the average of the log of the final vs initial abundance of all gRNAs per gene, and showed a distribution consistent with the expectation that the majority of the gRNAs do not have a major impact on cell viability. Experiments with evidence of random genetic drifts from the CRISPR scores distribution were excluded from the analyses. Initial hits, defined as kinase genes with targeting gRNAs consistently depleted in SF-mutant lines of all 3 genotypes, but not in the normal isogenic cells are being validated with individual gene knockout and small molecule inhibition. In parallel, we are setting up CRISPR screens in expandable HPCs (eHPCs) derived from iPSCs. The latter can be expanded in culture for several weeks and could enable screening of larger or even genome-wide gRNA libraries. DisclosuresNo relevant conflicts of interest to declare.

A “De Novo Leukemogenesis” iPSC Model Charts the Clonal Evolution of Acute Myeloid Leukemia

Acute Myeloid Leukemia (AML) develops through the stepwise acquisition of mutations by hematopoietic stem or progenitor cells in a process of clonal evolution, which drives disease initiation, progression and relapse. In recent years, large scale sequencing of patients with AML yielded nearly complete catalogues of the driver mutations, uncovered their temporal occurrences and classified AML subgroups based on mutational profiles. The next daunting challenge is to understand how these mutations contribute to the development of Myelodysplastic Syndrome (MDS) and AML and how the order of their acquisition impacts disease progression.To study the individual and cooperative effects of driver mutations in MDS and AML and model clonal evolution, we developed iPSC models by introducing mutations in a stepwise manner through serial CRISPR/Cas9-mediated gene editing. We selected two mutational paths, representing de novo AML and secondary AML from preexisting MDS, respectively: (a) the combination DNMT3AR882H, NPM1c and FLT3ITD, the most common 3-gene co-mutation in AML, associated with poor prognosis; and (b) the combination SRSF2P95L, ASXL1 C-terminus truncation and NRASG12D, which represents the “chromatin-spliceosome” AML genomic classification group. The normal iPSC line N-2.12, previously extensively characterized in terms of pluripotency, genetic integrity and hematopoietic differentiation potential, was used as the parental line. Each gene editing step entailed screening of single cell clones by RFLP analysis, confirmation of correct targeting and presence of an intact untargeted allele by DNA sequencing, a second round of single-cell cloning to ensure clonality and karyotyping to exclude chromosomal abnormalities. At least two clones from each step were subjected to hematopoietic differentiation to test for potential phenotypic discrepancies and one representative clone was selected for the next gene editing step.For the DNMT3AR882H, FLT3ITD, SRSF2P95L and NRASG12D mutations, CRISPR-mediated homology directed repair (HDR) was used. ASXL1mutant lines were generated by CRISPR/Cas9 targeting of the beginning of exon 12 of ASXL1 and selection of clones with frame-shifting indels. NPM1c was introduced with a dox-inducible lentiviral vector. We thus derived a panel of single, double and triple mutant clones representing each step of the clonal evolution of AML.Single mutant clones (DNMT3AR882H and ASXL1-truncated) had completely normal hematopoietic differentiation potential (similar to that of the parental normal line and other non-isogenic normal lines), which is not surprising given that both these mutations are also found in individuals with clonal hematopoiesis without any hematopoietic defects. The double mutant SRSF2P95L-ASXL1 cells showed moderately impaired early hematopoietic differentiation potential with colony-forming ability reduced to approximately 50% of normal. In contrast, both triple mutant lines (DNMT3AR882H-FLT3ITD-NPM1c and SRSF2P95L-ASXL1-NRASG12D) showed a more severe differentiation block with markedly reduced colony-forming ability and prolonged growth in culture for over 11 weeks, in striking contrast to normal iPSC-derived hematopoietic progenitor cells (HPCs) which completely arrest their proliferation after 4 weeks. Transplantation of SRSF2P95L-ASXL1-NRASG12DHPCs into NSG mice resulted in detectable engraftment after 9 weeks in all transplanted animals.In conclusion, we successfully employed CRISPR/Cas to introduce driver mutations into iPSCs in a stepwise manner to “de novo” reconstruct the development of AML. These panels of iPSCs capture the distinct mutational steps along the evolution of AML in a clonal state and isogenic conditions. They should enable mechanistic studies into the processes of leukemogenesis and the effects of specific mutations acting at distinct stages of this progression, their cooperation and the order by which they are acquired. They should also prove a powerful tool to investigate the minimal genetic requirements for leukemia development, the potential need for cooperating epigenetic insults and the effects of the cellular context in myeloid transformation. DisclosuresNo relevant conflicts of interest to declare.

Chronic myeloid leukaemia cells require the bone morphogenic protein pathway for cell cycle progression and self-renewal

Leukaemic stem cell (LSC) persistence remains a major obstacle to curing chronic myeloid leukaemia (CML). The bone morphogenic protein (BMP) pathway is deregulated in CML, with altered expression and response to the BMP ligands shown to impact on LSC expansion and behaviour. In this study, we determined whether alterations in the BMP pathway gene signature had any predictive value for therapeutic response by profiling 60 CML samples at diagnosis from the UK SPIRIT2 trial and correlating the data to treatment response using the 18-month follow-up data. There was significant deregulation of several genes involved in the BMP pathway with ACV1C, INHBA, SMAD7, SNAIL1 and SMURF2 showing differential expression in relation to response. Therapeutic targeting of CML cells using BMP receptor inhibitors, in combination with tyrosine kinase inhibitor (TKI), indicate a synergistic mode of action. Furthermore, dual treatment resulted in altered cell cycle gene transcription and irreversible cell cycle arrest, along with increased apoptosis compared to single agents. Targeting CML CD34+ cells with BMP receptor inhibitors resulted in fewer cell divisions, reduced numbers of CD34+ cells and colony formation when compared to normal donor CD34+ cells, both in the presence and absence of BMP4. In an induced pluripotent stem cell (iPSC) model generated from CD34+ hematopoietic cells, we demonstrate altered cell cycle profiles and dynamics of ALK expression in CML-iPSCs in the presence and absence of BMP4 stimulation, when compared to normal iPSC. Moreover, dual targeting with TKI and BMP inhibitor prevented the self-renewal of CML-iPSC and increased meso-endodermal differentiation. These findings indicate that transformed stem cells may be more reliant on BMP signalling than normal stem cells. These changes offer a therapeutic window in CML, with intervention using BMP inhibitors in combination with TKI having the potential to target LSC self-renewal and improve long-term outcome for patients.

Modeling APC mutagenesis and familial adenomatous polyposis using human iPS cells.

Mutations in the gene Adenomatous Polyposis Coli or APC appear in most sporadic cases of colorectal cancer and it is the most frequent mutation causing hereditary Familial Adenomatous Polyposis. The detailed molecular mechanism by which APC mutations predispose to the development of colorectal cancer is not completely understood. This is in part due to the lack of accessibility to appropriate models that recapitulate the early events associated with APC mediated intestinal transformation. We have established a novel platform utilizing human induced Pluripotent Stem cells or iPSC from normal or FAP-specific APC mutant individuals and evaluated the effect of the mutation in the cells before and after differentiation into intestinal organoids. In order to minimize genetic background effects, we also established an isogenic platform using TALEN-mediated gene editing. Comparison of normal and APC mutant iPSC revealed a significant defect in cell identity and polarity due to the presence of APC in heterozygosity as well as chromosomal aberrations including abnormal anaphases and centrosome numbers. Importantly, upon specification into intestinal progeny, APC heterozygosity was responsible for a major change in the transcriptional identity of the cells with dysregulation of key signaling pathways, including metabolic reprogramming, abnormal lipid metabolism and intestinal-specific cadherin expression. In conclusion, we have developed a novel iPSC/intestinal model of APC mutagenesis and provide strong evidence that APC in heterozygosity imparts a clear phenotypic and molecular defect, affecting basic cellular functions and integrity, providing novel insights in the earlier events of APC-mediated tumorigenesis.

The Activated TGFβ Pathway in Shwachman Diamond Syndrome Impairs Hematopoiesis and Is Down-Regulated By Deletion of 7q

Monosomy 7 or deletion of 7q (del(7q)) are common cytogenetic abnormalities in pediatric MDS. Monosomy 7/del(7q) frequently arises in the context of inherited bone marrow failure (BMF) syndromes such as Shwachman Diamond Syndrome (SDS), an autosomal recessive disorder caused by biallelic mutations in the SBDS gene. Monosomy 7/del(7q) is associated with high grade MDS and a high risk of malignant transformation to acute myelogenous leukemia, a major cause of morbidity and mortality for patients with inherited BMF. The basis for this propensity to develop monosomy 7/del(7q) remains unclear. Whether monosomy 7/del(7q) functions as a driver of MDS, or is merely an associated marker of clonal progression in BMF, remains a critical question. The aim of this study is to investigate the molecular consequences of del(7q) in the context of BMF with the goal of developing more effective treatments.The lack of synteny between murine and human chromosome 7 has posed a major barrier to modeling monosomy 7/del(7q). Therefore, we utilized SDS patient-derived induced pluripotent stem cells (SDS-iPSC), which recapitulate the hematopoietic defects of this BMF syndrome. Independent SDS-iPSC lines were derived from two patients with SDS. Proliferationof SDS-iPSCs was reduced relative to wild type controls without a concomitant increase in cell death. Compared to normal iPSC controls, SDS iPSC-derived CD34+ cells exhibited reduced hematopoietic differentiation to CD45+ cells, decreased erythroid and myeloid hematopoietic progenitor colony formation in methylcellulose, and impaired terminal myeloid differentiation.To study the molecular and biologic effects of del(7q), a deletion of the MDS-associated region of the long arm of chromosome 7 was genomically engineered using a previously published modified Cre-Lox approach. The deletion of 7q in SDS iPSCs spanned band q11.23 to q36.3. The SDS-del(7q) iPSCs expressed stem cell markers and formed teratomas as efficiently as their isogenic SDS iPSC controls.Deletion of 7q failed to confer a relative growth advantage even within the context of BMF. Indeed, proliferation of the SDS-del(7q) iPSCs was reduced below that of both the isogenic SDS iPSCs and normal controls without an increase in cell death. To investigate the effect of del(7q) on hematopoiesis, iPSC-derived CD34+ cells were assayed for their ability to undergo hematopoietic differentiation. SDS-del(7q) CD34+ cells demonstrated reduced differentiation to CD45+ cells, reduced methylcellulose hematopoietic progenitor colony formation and impaired terminal myeloid differentiation compared with isogenic SDS CD34+ cells. These data demonstrate that deletion of 7q fails to confer a relative growth advantage relative to isogenic SDS iPSCs and results in a further impairment of hematopoiesis.To gain insight into the mechanisms of del7q-associated clonal evolution in SDS, we performed RNA sequencing (RNAseq) of SDS and SDS-del(7q) iPSC. Expression of TGFβ pathways and their downstream targets were reduced in SDS del(7q) iPSCs compared to isogenic SDS iPSC controls. This observation was intriguing because single cell RNAseq analysis of primary SDS bone marrow cells revealed that TGFβ pathway is hyperactivated in SDS. Additionally, a quantitative DNA aptamer-based proteomic analysis demonstrated elevated levels of TGFβ ligands in plasma from six SDS patients compared to 6 age-matched controls. Western blot analysis showed increased P-SMAD2 levels in SDS iPSCs compared to SDS-del(7q) and normal controls, while total levels of SMAD2 were unchanged. Pharmacological targeting of TFGβ with small molecule inhibitors resulted in selective improvement of SDS hematopoietic colony formation without stimulating outgrowth of the isogenic SDS-del(7q) cells. Although del(7q) does not seem to confer an advantage in growth or hematopoiesis in vitro, we are testing whether the deletion of 7q might confer relative resistance to elevated TGFβ levels in vivo, resulting in an overall survival advantage of the del7q clone.These results demonstrate that the TGFβ pathway is activated in SDS, and that deletion of 7q reverses the TGFβ pathway hyperactivation in SDS. Inhibition of TGFβ selectively rescues hematopoiesis in SDS but not in isogenic del7q cells, suggesting a potential strategy to treat bone marrow failure without stimulating del7q clonal outgrowth. DisclosuresStegmaier:Novartis: Consultancy, Research Funding. Shimamura:TransCellular Therapeutics: Other: Spouse is majority shareholder.

Generation and characterization of human iPSC lines SANi001-A and SANi002-A from mobilized peripheral blood derived megakaryoblasts

Mobilized peripheral blood (MPB) CD34+ cells were differentiated to CD34+/CD41+ megakaryoblasts. Cells were sorted to obtain a pure megakaryoblast population which was reprogrammed with a hOKSM self-silencing polycistronic lentiviral vector. Resulting iPSC showed normal karyotype and expression of pluripotency associated markers and in vitro spontaneous differentiation towards the 3 germ layers confirmed pluripotency of iPSC lines. Besides normal iPSC applications, these lines can be used as a control line for other megakaryoid origin iPSC and could be applied for epigenetic based research.

Abstract 1936: Epigenetic challenges in derivation of the first cell-based model of Beckwith-Wiedemann Syndrome

Abstract Beckwith-Wiedemann Syndrome (BWS) is a cancer predisposition syndrome that affects at least 1 in 10,500 children. Up to 25% of children with BWS develop tumors, primarily Wilms tumor and hepatoblastoma. BWS is due to genetic or epigenetic changes that affect imprinted loci on chromosome 11 and these same changes are also found in other types of cancer. There are no cell-based models of BWS and most mouse models do not recapitulate the tumor phenotype. To understand more about the mechanisms leading to tumor formation in BWS, we developed the first human cell-based model of BWS. Human induced pluripotent stem cell (iPSC) models are commonly used to study disease mechanisms in tissue types that are not normally accessible for study. In the case of BWS, we plan to use such models to study how the genetic and epigenetic changes in BWS lead to tumor formation in hepatic and renal cells. Using skin fibroblasts from four BWS patients, we derived the first iPSC models of BWS. Prior to iPSC derivation, we characterized different tissues available from these four patients, and demonstrated genetic mosaicism in different tissue types, including blood, skin, and pancreas. During the derivation process, we demonstrated that both normal and BWS iPSC lines could be derived from the same patient fibroblast sample and the number of clones of each type from each sample approximated the initial level of mosaicism in the original sample. For each patient, these lines are isogenic except for the BWS critical region. The BWS and isogenic normal iPSCs were characterized for pluripotency markers and demonstrated to have normal karyotypes. Following this analysis, BWS and isogenic normal lines were characterized extensively for DNA methylation at specific imprinted loci in both early passage and extended culture. Methylation analysis was performed by both pyrosequencing and COBRA assays. Methylation was maintained at some imprinted loci but not at others in extended culture. Importantly, relatively stable methylation levels were observed at the BWS critical imprinted regions (H19/IGF2 and KvDMR), regardless of methods of reprogramming, indicating a relatively stable state of DNA methylation in this region. Additionally, normal methylation was seen at the SNRPN locus. In contrast, another imprinted locus, IG/MEG3, displayed abnormal hypermethylation in iPSCs. These data indicate that reprogramming and extended culture of iPSCs can affect stability of DNA methylation at certain imprinted loci. Therefore caution should be used in interpreting studies using iPSCs as these aberrant methylation states at imprinted loci can affect the downstream functionality of iPSC models. BWS iPSCs and isogenic controls with normal methylation will be used for further study of the mechanism of tumor formation in BWS. Note: This abstract was not presented at the meeting. Citation Format: Stella K. Hur, Joanne L. Thorvaldsen, Suhee Chang, Carolyn Lye, Alice Yu, Monserrat C. Anguera, Marisa S. Bartolomei, Jennifer M. Kalish. Epigenetic challenges in derivation of the first cell-based model of Beckwith-Wiedemann Syndrome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1936. doi:10.1158/1538-7445.AM2017-1936

199 EFFECTS OF REPROGRAMMING-CONDITIONED MEDIUM ON ULTRAVIOLET RAY A–DAMAGED HUMAN DERMAL FIBROBLASTS

Ultraviolet ray A (UVA) is an electromagnetic light with a long wavelength from the sun. The penetration of UVA deep into the human dermis causes changes in cells, such as DNA fragmentation, apoptosis, and senescence, eventually leading a decline of proliferation and wound-healing ability. These changes induced by UVA exposure are similar to those seen in the process of stem cell differentiation. We postulated that the condition that reverses cellular differentiation may alleviate the UVA-induced damage in skin cells. Human dermal fibroblasts (HDF) could be reprogrammed to induced pluripotent stem cells (iPSC). Conditioned medium (CM) was prepared during the process of iPSC reprogramming (referred to as Repro-CM). The UVA-irradiated HDF were cultured in Repro-CM for 24 h. In comparison with CM prepared from the culture of normal HDF and iPSC (referred to as HDF-CM and iPSC-CM, respectively), effects of Repro-CM on UVA-irradiated cells were investigated. Viability, wound-healing ability, apoptosis, and senescence of HDF were analysed by WST-1 assay, scratch assay, Annexin V assay, and senescence-associated β-galactosidase assay, respectively. Upon recovering from the UVA-induced damage, viability and wound-healing ability of HDF were significantly different (P < 0.05) among the treatments in the order of Repro-, HDF-, and iPSC-CM. In the same context, apoptosis and senescence were significantly different (P < 0.05) in the order of iPSC-, HDF-, and Repro-CM. Interestingly, iPSC-CM did not substantially ameliorate UVA-induced damage, suggesting that the conditions optimized to pluripotent stem cells may not be suitable for the recovery from damage in terminally differentiated cells, such as fibroblasts. The RNA-seq analysis was performed to assess the genome-wide transcriptional profile in the process of recovery. Repro- and HDF-CM were categorized more closely than iPSC-CM in hierarchical cluster analysis. In comparison with iPSC-CM, the up-regulated genes by Repro-CM treatment were related to regulation of cell proliferation and cell metabolism, whereas down-regulated genes were related to antiapoptosis and response to stimulation of chemical and organic substances. Overall, providing an environment of reprogramming, as shown by Repro-CM in the present study, may assist recovery of HDF from UVA-induced damage. The results of the study may be applicable in developing pharmaceuticals to treat aging and wrinkling of the skin caused by UVA irradiation.

Ips Technology Revealed the Genetic and Functional Diversity Present in a Secondary AML Patient

Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal stem cell diseases characterized by inefficient hematopoiesis and risk of progression to acute myeloid leukemia with poor prognosis. Although massive parallel sequencing studies have revealed a number of genomic alterations associated with myelodysplastic syndromes (MDS), the relationships between genetic architecture and clonal evolution in MDS remain poorly understood, mainly due to a difficulty in the ex vivo culture of primary MDS cells and a lack of good animal models. Induced pluripotent stem cells (iPSCs) from MDS patients are expected to provide a new platform for elucidation of the pathogenesis of MDS. We used reprogramming technology and transplantation into immunodeficient mice to functionally dissect the genetically defined subclones of secondary acute myeloid leukemia (AML) evolving from MDS (MDS/sAML).We attempted to generate iPSCs from bone marrow and peripheral blood mononuclear cells of a MDS/sAML patient with normal karyotype and multiple FLT3-internal tandem duplication (ITD) mutations, utilizing episomal methods. We successfully established more than 30 iPSC lines from sAML clones (sAML-iPSC) and 4 normal iPSC lines from normal clones at the same time. sAML-iPSC lines displayed characteristic morphology and expressed pluripotent stem cell markers at the levels comparable to those in isogenic normal iPSC lines and ES cell lines. Five randomly selected sAML-iPSC lines formed teratomas. Most of the sAML-iPSC lines retained normal karyotype even after 30 passages. About half of the sAML-iPSC lines harbored one out of four types of FLT3-ITDs identical to the primary sAML cells, reflecting the mosaicism of the FLT3-ITDs in the patient's sAML cells. Whole exome sequencing and SNP-CGH analysis revealed that all sAML-iPSC lines shared the chromosome 11p uniparental disomy (UPD) and 14 somatic single nucleotide variants (SNVs) identical to those of the primary sAML cells, indicating that these iPSC lines are derived from clonal sAML cells. Additionally, most of the sAML-iPSC lines had a frameshift mutation of CEBPA. Unexpectedly, 2 of the sAML-iPSC lines without any FLT3-ITD mutations had a FLT3-D835Y mutation, which was only detected by droplet digital PCR (ddPCR) at extremely low levels in the primary bone marrow sample.Hematopoietic differentiation efficiency of sAML-iPSC lines was comparable to that of isogenic normal iPSC lines. However, CD34+CD38-CD45+lineage- hematopoietic stem and progenitor cells (HSPCs) from sAML-iPSC lines displayed two to three times enhanced colony formation ability in myeloid-conditioning semifluid medium. Colonies formed by sAML-iPSC-derived HSPCs were predominantly composed of immature myeloblasts and could be replated for at least five rounds. Both FLT3-ITD- and FLT3-D835Y-positive sAML-iPSC-derived hematopoietic progenitor cells (HPCs) exhibited cytokine-independent growth but only FLT3-ITD-positive sAML cell lines retained in vitro sensitivity to AC220. To functionally evaluate the subclonal diversity in vivo, we performed xenotransplantation of HPCs from several sAML-iPSC lines into sublethally irradiated NSG mice. HPCs from all tested sAML-iPSC lines engrafted at 13 to 15 weeks after transplant. HPCs from FLT3-ITD-positive sAML-iPSC lines (n=5) displayed a higher ability to engraft and induced more severe sAML-like disease in recipient mice than those from FLT3-wild-type (wt) sAML-iPSC lines (n=4) (median spleen weight, 145.4 mg vs 55.7 mg; P < .05). Survival of mice injected with FLT3-ITD-positive sAML-iPSC-derived HPCs was shorter than those injected with FLT3-wt sAML-iPSC-derived HPCs (194 days vs 361 days; P< .001). To confirm the transformation properties of the FLT3-ITDs in this MDS/sAML patient, we transduced FLT3-wt sAML-iPSC-derived HPCs with retrovirus expressing a FLT3-ITD and transplanted the HPCs into recipient mice. FLT3-ITD-transduced recipients developed an aggressive sAML-like disease in 100% of recipient mice with shorter latency around 6 to 10 weeks, as evidenced by marked splenomegaly and massive expansion of immature myeloid cells in bone marrow and spleen.Our data suggest that this iPSC-based system could be useful for analysing clonal architecture and biological feature of subclones of MDS/sAML to identify candidate genes responsible for initiation and progression of MDS. DisclosuresTakaori-Kondo:Shionogi: Research Funding; Toyama Chemical: Research Funding; Mochida Pharmaceutical: Research Funding; Eisai: Research Funding; Cognano: Research Funding; Alexion Pharmaceuticals: Research Funding; Takeda Pharmaceutical: Research Funding; Astellas Pharma: Research Funding; Kyowa Kirin: Research Funding; Chugai Pharmaceutical: Research Funding; Pfizer: Research Funding; Janssen Pharmaceuticals: Speakers Bureau; Merck Sharp and Dohme: Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau. Yamanaka:iPS Academia Japan: Consultancy.

Establishment and gene expression analysis of disease-derived induced pluripotent stem cells of scleroderma

BackgroundWe recently generated induced pluripotent stem cells (iPSCs) from cultured dermal fibroblasts of systemic sclerosis (SSc-iPSC) to study the disease mechanisms. ObjectiveIn the present study, we have performed gene expression analysis using cultured SSc dermal fibroblasts, SSc-iPSC, and fibroblasts re-differentiated from SSc-iPSC (SSc-iPSC-FB). MethodsmRNA and protein levels of collagen and integrins were analyzed using PCR array, PCR, immunoblotting, and immunofluorescence. ResultsWe compared expression pattern of TGF-β-related genes between normal iPSC (NS-iPSC) and SSc-iPSC by PCR array, and found constitutive and significant down-regulation of S100A8, Smad6, and TGF-β2 in SSc-iPSC.The expression of these genes was not altered in cultured SSc fibroblasts or SSc-iPSC-FB compared to NS fibroblasts or NS-iPSC-FB, respectively. On the other hand, the expression of collagen, integrin α and β was up-regulated in SSc fibroblasts, while SSc-iPSC-FB showed normalized levels of collagen and integrin β. ConclusionsSo far, there have been no reports investigating disease-derived iPSCs of SSc. Our results suggest that S100A8, Smad6, and TGF-β2 may be the key molecules of this disease. On the other hand, the normalization of collagen and integrins by iPSC reprogramming suggests that epigenetic modifications of genes may play a role in the mechanism of collagen accumulation seen in SSc fibroblasts, and that gene reprogramming may become novel therapeutic approach.As the limitation of this study, we established only one iPSC line from each patient, which may not be enough to discuss disease-specific phenotypes. Larger studies including increased number of iPSC lines are needed in the future.

Abstract 2483: Generation of patient-specific and genome-edited MEN2A patient-derived induced pluripotent stem cells as novel tools for drug screening

Abstract Introduction MEN2A is a cancer-predisposing syndrome that affects patients with germline RET mutations. Animal models do not faithfully recapitulate the full clinical spectrum of the disease. Moreover, it has been suggested that primitive malignant stem cells could be at the origin of resistances and relapses in patients treated with standard chemotherapy. In order to modelling MEN2A malignant primitive stem cells, we have induced to pluripotency MEN2A peripheral blood mononuclear cells from patients who developed medullary thyroid carcinoma harboring “high risk” or “moderate risk” RET mutation. Methods PBMC from patients with germline RETC620R and RetC634Y mutations were reprogrammed into pluripotent stem cells using Sendai-virus mediated Oct4, Sox2, Klf4 and c-Myc gene transfer. An isogenic IPSC line was generated by genome editing, using CRISPR-Cas9 to correct the “high risk” RETC634Y mutation. Hallmarks of pluripotency, genotype and phenotypes were characterized. Results Pluripotency behaviour of each IPSC line was confirmed in vitro (EBs, and pluripotent markers) and in vivo (teratoma assay in immunodeficient mice). Genome edition of the RETC634Y iPSC led to the generation of the RETY634C isogenic control iPSC. Ret expression was confirmed on both RETC634Y and RETY634C. In order to determine the accuracy and absence of off-target effects, we have performed a whole exome sequencing of both RET-mutated and RET-corrected IPSCs. These experiments revealed some off target effects of the CRISPR-mediated gene edition. Gene-array datas revealed an increased expression of neuronal development-related set of genes in RETC634Y iPSC, as compared to RETY634C control, that may account for the development of certain MEN2A features. Treatment of IPSC with vandetanib, a Ret inhibitor, led to a decrease of iPSC colonies formation in RETC634Y mutated iPSC compared to RETY634C, suggesting that RET-iPSC could serve as a plateform drug screening model. The comprehensive pathological assessment of IPSC RET-mutated and corrected derived-teratoma did not reveal, at this point, any neuroendocrine tumor-reminiscent structure. Conclusion We report here the first MEN2A-iPSC from “high risk” RETC634Y iPSC and its genome-edited isogenic normal IPSC, with their genomic profiling by whole exome sequencing. These well caracterized RET-IPSC will allow to develop cell-based assays in high throughput screening for drug discovery and to model MEN2A. Citation Format: Julien Hadoux, Christophe Desterke, Olivier Féraud, Mathieu Guibert, Roberta Francesca De Rose, Paule Opolon, Dominique Divers, Emilie Gobbo, Frank Griscelli, Martin Schlumberger, Annelise Bennaceur-Griscelli, Ali Turhan. Generation of patient-specific and genome-edited MEN2A patient-derived induced pluripotent stem cells as novel tools for drug screening. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2483.

Modeling Keratoconus Using Induced Pluripotent Stem Cells.

PurposeTo model keratoconus (KC) using induced pluripotent stem cells (iPSC) generated from fibroblasts of both KC and normal human corneal stroma by a viral method.MethodsBoth normal and KC corneal fibroblasts from four human donors were reprogramed directly by delivering reprogramming factors in a single virus using 2A “self-cleaving” peptides, using a single polycistronic lentiviral vector coexpressing four transcription factors (Oct 4, Sox2, Klf4, and Myc) to yield iPSC. These iPS cells were characterized by immunofluorescence detection using of stem cell markers (SSEA4, Oct4, and Sox2). The mRNA sequencing was performed and the datasets were analyzed using ingenuity pathways analysis (IPA) software.ResultsThe generated stem cell-like clones expressed the pluripotency markers, SSEA4, Oct4, Sox2, Tra-1-60, and also expressed pax6. Our transcriptome analysis showed 4300 genes, which had 2-fold change and 870 genes with a q-value of <0.05 in keratoconus iPSC compared to normal iPSC. One of the genes that showed difference in KC iPSC was FGFR2 (down-regulated by 2.4 fold), an upstream target of Pi3-Kinase pathway, was further validated in keratoconus corneal sections and also KC iPSC-derived keratocytes (down regulated by 2.0-fold). Both normal and KC-derived keratocytes expressed keratocan, signature marker for keratocytes. KC iPSC-derived keratocytes showed adverse growth and proliferation and was further confirmed by using Ly2924002, a PI3k inhibitor, which severely affected the growth and differentiation in normal iPSC.ConclusionsBased on our result, we propose a model for KC in which inhibition FGFR2-Pi3-Kinase pathway affects the AKT phosphorylation, and thus affecting the keratocytes survival signals. This inhibition of the survival signals could be a potential mechanism for the KC-specific decreased cell survival and apoptosis of keratocytes.

A Library of Sickle Cell Anemia Induced Pluripotent Stem Cells of Diverse Haplotypes and Ethnicities

The mutation causing sickle cell anemia (rs334, GAG-GTG, glu6val) had several independent origins in Africa, the Middle East and India and spread throughout parts of the world by wars, slave trading and population migrations. The genetic background upon which the HbS mutation occurred, or the β-globin gene (HBB) haplotype, is associated with differences in the phenotype of this disease and the ability of affected individuals to synthesize fetal hemoglobin (HbF). The main modifier of the disease phenotype is the level of HbF in the blood of affected individuals. HbF inhibits the polymerization of HbS, the proximate cause of disease pathophysiology. As part of the NHLBI NextGen consortium (U01HL107443) we established a library of induced pluripotent stem cells (iPSC) from patients with sickle cell anemia of diverse HBB haplotypes and HbF phenotypes. The purpose of establishing this library was to allow genetic studies of globin gene expression during the erythroid differentiation of iPSC of diverse genotypes. During these studies we have implemented an efficient and highly reproducible platform for the production of large numbers of sickle cell anemia-specific iPSC, derived and characterized a novel in vitro system for the production of an unlimited supply of erythroid lineage cells from the directed differentiation of normal and disease-specific iPSC and used this system to recapitulate erythroid-lineage ontogeny in vitro with the sequential development of primitive and definitive erythropoiesis, accompanied by the appropriate expression of stage-specific globin genes. We have recently finished whole genome DNA and RNA sequencing analysis in some of these lines aimed at identifying developmental gene expression profile differences between erythroid precursors that produce primarily HbF and those that produce primarily HbA or HbS as part of our search for novel HbF genetic modifiers associated with markedly elevated HbF levels found in sickle cell anemia patients naturally, or in response to hydroxyurea treatment. Furthermore, our labs are also focusing on using a CRISPR-based gene editing platform to study the effect of novel HbF genetic modifiers and explore globin switching. Cell lines established are shown in the table.Table 1Number of subjects recruited to date98Number of subjects with iPSC lines established56Average number of iPSC lines per subject3 (total of 158 lines generated)Quality control status of iPSC linesAll lines are expanded and banked, mycoplasma free, express pluripotency markersSubjects with target cells differentiated (erythrocytes)25Samples have been collected on African American patients with sickle cell anemia with diverse HBB haplotypes, predominantly homozygotes and compound heterozygotes for the Benin and Bantu haplotypes, Saudi Arabian patients with the Arab-Indian haplotype and the Saudi Benin haplotype that is characterized by HbF levels about twice as high as in African Benin haplotype patients and from Brazilian patients who are predominantly homozygotes for the Bantu haplotype that typically is associated with the lowest HbF of all HBB haplotypes. This iPSC-based library and the data associated with it represents a valuable readily available resource for the sickle cell research community and all the generated lines will be available for distribution early in 2016 through WiCell. DisclosuresNo relevant conflicts of interest to declare.

Differential Sensitivity to JAK Inhibitory Drugs by Isogenic Human Erythroblasts and Hematopoietic Progenitors Generated from Patient-Specific Induced Pluripotent Stem Cells

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 CD34+ 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.

An iPSC-Based Model Of MDS For Phenotype-Driven Gene and Drug Discovery

Despite past and ongoing research efforts, the pathogenetic mechanisms of MDS remain far from understood sufficiently to point to single genes or molecular pathways as putative targets for generation of better mouse models or drug development. In lack of clear targets, the manipulation of disease-associated phenotypes may at present be the best opportunity for disease study and therapeutic intervention. Furthermore, because cellular phenotypes are more proximal to molecular disease mechanisms than phenotypes seen at the level of the tissue or organism (i.e. the murine or human hematopoietic system), they may provide more sensitive and relevant readouts of the disease process.With the advent of patient-specific induced pluripotent stem cells (iPSCs), disease models based on cellular phenotypes (“disease-in-a-dish”) can now be developed and their unique properties promise to revolutionize disease study and drug development. Unlimited cell numbers of biologically relevant cells can be obtained relatively easily and cost-effectively. Here we present a new MDS model based on patient-specific iPSCs and its use in two phenotype-driven screens: (a) a focused genetic screen and (b) a high-throughput chemical screen.We have derived multiple MDS-iPSC lines with deletions of chromosomes 7q or 20q (characteristic chromosomal deletions that we are using as markers of iPSCs derived from the MDS clone) from bone marrow (BM) of 3 patients with MDS and 1 patient with sAML. We also derived isogenic karyotypically normal iPSC lines in parallel from the same BM samples. We identified two cellular phenotypes specific to the MDS-iPSCs: decreased proliferation rate and decreased potential for pan-hematopoietic differentiation and reduced clonogenic capacity of their hematopoietic progeny. These phenotypes are consistent across multiple iPSC lines from different patients and absent from their isogenic normal iPSC controls. They are reminiscent of the behavior of ex vivo cultured primary MDS BM cells and are therefore likely to be relevant to the disease process. Furthermore, they are rescued by spontaneous compensation for chr7q dosage through acquisition of an extra chr7 and recapitulated by the engineering of artificial chr7q deletions in normal iPSCs.To identify critical MDS gene(s) on chr7q, we performed a screen of 62 candidate haploinsufficient genes (with reduced expression by at least 1.5-fold in our del(7q)-iPSCs compared to their isogenic normal iPSCs) for rescue of the proliferation and/or hematopoietic differentiation phenotype. We constructed a lentiviral library of all candidate ORFs (and 14 additional alternative transcripts) linked to eGFP through a P2A peptide and each tagged with a unique 4-nt barcode sequence to its 3' UTR. The library was packaged as a pool and transduced into two different del(7q)-iPSC lines. The cells were passaged for 16 weeks and gDNA was isolated every 2 weeks. In parallel, the cells were differentiated along the hematopoietic lineage and gDNA was isolated from CD45+ cells FACS-sorted on day 15 of differentiation. High-throughput sequencing of the barcodes identified 5 ORFs that became enriched in undifferentiated iPSCs over time (rescue of proliferation) and 4 ORFs enriched in sorted CD45+hematopoietic progenitors (rescue of hematopoietic differentiation), 2 of which overlapped. All 9 hits are being further validated in more focused screens.Second, we used the same platform for a high-throughput small molecule screen. We optimized the plating conditions and densities for a 384-well format using a luminescent cell viability assay and conducted a screen of 2000 compounds comprising known drugs, natural products, and other bioactives and chemicals, in an MDS-iPSC line (2.13) and its isogenic normal control (2.8). Primary hits were defined as compounds that enhanced the growth of the MDS-iPSC line, but not of the control normal iPSC line in a compound dose-dependent manner. 38 primary hits were retested in a dose-response survival assay in one additional MDS- (2.A1-3), one additional isogenic normal iPSC line (2.12) and one sAML iPSC line over 8 concentrations and 12 compounds were prioritized for further studies.Our data highlight the potential of this new iPSC-based model of MDS for screens to identify genes or compounds that affect cellular phenotypes by acting on previously undefined targets. Disclosures:No relevant conflicts of interest to declare.

Myelopoiesis From Induced Pluripotent Stem Cells Reveals The Role Of Elastase Activity In The Pathogenesis Of Severe Congenital Neutropenia

Severe congenital neutropenia (SCN) is a hereditary neutropenia characterized by granulocytic precursor differentiation arrest in which the pathogenic mechanism is poorly understood. Over half of SCN patients associate with autosomal dominant point mutations in the gene encoding neutrophil elastase (ELANE). High-dose G-CSF therapy succeeds in increasing neutrophil counts in many SCN patients, but the molecular explanation for high dose G-CSF rescue is unknown. Mutations in the G-CSF receptor (CSF3R) associated with a dramatic risk for transformation provides a cautionary note for high dose G-CSF treatment. The pathogenic mechanism of ELANE mutations is incompletely understood due to the lack of disease recapitulation in murine models of ELANE-mutant hematopoiesis and the difficulty to obtain primary, relevant myeloid cell populations from patients. Using induced pluripotent stem cell (iPSC) derived myelopoietic cultures from peripheral blood mononuclear cells of patients with ELANE exon 3 point mutations (Q97P and I118N), we designed experiments to recapitulate G-CSF signaling in SCN myelopoiesis. The patient-derived iPSC lines were characteristically similar to human embryonic stem cell lines and normal blood-derived iPSC lines with expression of SSEA-4, Tra-1-60, Tra-1-81, and CD9 pluripotency markers &gt;85% in all lines as determined by FACS analysis at passage 10-15. All iPSC retained a normal karyotype and ELANE locus mutations of the original specimens. Both SCN iPSC lines and control iPSC lines (normal and immune-mediated congenital neutropenia) show functional G-CSF (50 ng/mL)-induced differentiation into the myeloid lineage. However, G-CSF failed to induce terminal granulocytic differentiation of SCN iPSC derived myeloid progenitors resulting in differentiation arrest at the promyelocyte/myelocyte stage. The hematopoietic cells derived from SCN iPSC line show reduced frequency of CFU-G and CFU-GM (CFU-G+CFU-GM, 22±1.15 in 104 embryoid body cells from non SCN iPSC vs 4±2 in 104 embryoid body cells from ELANE mutated SCN iPSC). Moreover, SCN iPSC promyelocytes show significantly increased apoptosis over controls as determined by Annexin-V analysis (31% Annexin-V+ in SCN iPSC derived promyelocyte vs 3% in control iPSC). Surviving SCN promyelocytes showed decreased reactive oxygen species activity and no phagocytic activity. In contrast, suprapharmacological doses of G-CSF (1000 ng/mL) rescued SCN iPSC-derived differentiation. It has been shown that NE, expressed in promyelocytes, can cleave recombinant G-CSF and CSF3R. We evaluated the possibility that inadequate G-CSF signaling in SCN iPSC was due to abnormal elastase activity. To this end, we supplemented 50 ng/mL G-CSF cultures with a specific, cell-permeant neutrophil elastase inhibitor (Sivelastat, 230 nM) to find rescued granulocyte differentiation arrest (31% neutrophil in myeloid differentiation culture of SCN iPSC derived hematopoietic progenitors in presence of Sivelastat vs 2% mature neutrophils without Sivelastat). In conclusion, SCN modeling through patient iPSC derived myelopoiesis and G-CSF driven granulocytic recapitulation unveil a mechanism of resistance to G-CSF therapy with important translational implications for therapy. Combination of low-dose G-CSF and NE inhibitory therapy may result in an efficacious, safer approach in SCN therapy. Disclosures: No relevant conflicts of interest to declare.

Chromosome 7q Hemizygosity Recapitulates MDS-Related Cellular Phenotypes In Genetically Engineered Human Pluripotent Stem Cells

Loss of the entire or part of one copy of chromosome 7 [del(7/7q)] is a recurrent cytogenetic abnormality in MDS. Its strong association with previous exposure to alkylating agents, consistently poor response to therapy and discrete gene expression profile strongly suggest that del(7/7q)-MDS is a distinct disease in the MDS spectrum whose pathogenesis is intimately linked to loss of chr7q genetic material. Understanding the role of chr7q loss in cell biology can provide key insights into the pathogenesis of MDS and leukemogenesis.Narrowing down the responsible region on chr7q presents a challenge that has proved intractable with existing approaches. Chr7q deletions physically mapped in large cohorts of patients are typically very large and dispersed along most of the length of chr7q. Modeling in the mouse is problematic for reasons of synteny. Deletion of the syntenic region of one commonly deleted region (7q22 CDR) failed to demonstrate a phenotype.Several lines of evidence point to haploinsufficiency of chr7q genetic material – rather than a 2-hit model – as the underlying mechanism in del(7q)-MDS. No inactivating mutations of candidate 7q genes have been detected by resequencing the remaining allele and haploinsufficiency of coding and miRNA genes has been strongly linked to the pathogenesis of del(5q)-MDS. Haploinsufficiency can only be assessed through functional studies (and not genomic technologies), but these are currently hindered by the lack of a clearly described del(7q)-associated phenotype.With recent advances in human pluripotent stem cell (hPSC) research and genetic engineering technologies, reverse human genetics in an isogenic setting by disruption of genomic elements into their cognate genomic and cellular context - hitherto unthinkable for the human genome – are now a realistic prospect. To determine the impact of hemizygous chr7q loss on the cellular phenotype, we have generated isogenic del(7q)- and normal hPSCs by engineering deletions spanning variable overlapping regions encompassing the entire length of chr7q using adeno-associated virus (AAV)-mediated gene targeting combined with Cre-lox technology. Specifically, we targeted two inverted loxP sites together with a positive (puro) and a negative (HSVtk) selection marker in a near-telomeric region of chromosome 7q (7q36.3) into the H1 hESC line, as well as a karyotypically normal iPSC line (line 2-12) derived from BMMCs of a patient with del(7q)-MDS. Following transient expression of Cre recombinase and ganciclovir selection, clones were screened by qPCR probing different regions along the length of chr7. Four H1-derived and four 2-12-derived clones were selected following screening of 24 and 34 clones, respectively, and after excluding clones with additional chromosomal abnormalities by karyotyping and the exact extent of their chr7q deletions was mapped by aCGH.We focused our phenotypic characterization on two cellular phenotypes that we recently reported in del(7q)-iPSCs derived from MDS BMMCs: cell proliferation and in vitro hematopoietic differentiation potential. 7 of the 8 clones harboring deletions spanning variable lengths along the entire chr7q had a lower (by ½ log) proliferation rate than their corresponding isogenic parental lines. All these clones also exhibited a markedly reduced differentiation potential along all hematopoietic lineages and almost absent clonogenic capacity in methylcellulose. These cellular phenotypes are highly similar to those we find in our del(7q)-MDS-iPSCs. Notably, one of the eight clones (2-12.Cre-44), harboring a smaller deletion spanning 7q11.21-7q.31.1 retained comparable proliferation and differentiation capacity to that of normal isogenic and non-isogenic hPSCs.In conclusion, our results demonstrate that hemizygous loss of chr7q material recapitulates the cellular phenotypes of impaired proliferation and hematopoietic differentiation that we find in del(7q)-MDS-iPSCs, supporting a haploinsufficiency pathogenesis of del(7q)-MDS. Correlation of the phenotypes with the boundaries of chr7q deletions in our collection of hESC and iPSC clones points to a region spanning 7q31.1-7q36.1 as the critical region in del(7q)-MDS. Further studies in additional clones harboring smaller chr7q deletions will further narrow down the responsible region and guide prioritization of candidate genes. Disclosures:No relevant conflicts of interest to declare.

Abstract 262: Derivation of Duchenne Muscular Dystrophy Disease Specific Cardiomyocytes From Patient Urine

Introduction: Human somatic cells can be reprogrammed into primitive stem cells, termed induced pluripotent stem cells (iPSCs). These iPSCs can be extensively expanded in vitro and differentiated into multiple functional cell types, enabling faithful preservation of individual’s genotype and large scale production of disease targeted cellular components. These unique cellular reagents thus hold tremendous potential in disease mechanism study, drugs screening and cell replacement therapy. Due to the genetic mutation of the protein dystrophin, many DMD patients develop fatal cardiomyopathy with no effective treatment. The underlying pathogenesis has not been fully elucidated. Hypothesis: We tested the hypothesis that iPSCs could be generated from DMD patients’ urine samples and differentiated into cardiomyocytes, recapitulating the dystrophic phenotype. Methods: iPSCs generation was achieved by introducing a lentiviral vector expressing Oct4, Sox2, c-Myc and Klf4 into cells derived from patient’s (n=1) and healthy volunteers’ (n=3) urine. Cardiomyocytes were derived by sequentially treating iPSCs with GSK3 inhibitor CHIR99021 and Wnt inhibitor IWP4. Differentiated cardiomyocytes were subjected to calcium imaging, electrophysiology recording, Polymerase Chain Reaction (PCR) analysis, and immunostaining. Results: iPSCs were efficiently generated from human urine samples and further forced to differentiate into contracting cardiomyocytes. PCR analysis and immunostaining confirmed the expression of a panel of cardiac markers. Both normal and patient iPSC derived cardiomyocytes exhibited spontaneous and field stimulated calcium transients (up to 2Hz), as well as action potentials with ventricular-like and nodal-like characteristics. Anti-dystrophin antibodies stained normal iPSC-derived cardiomyocyte membranes but did not react against DMD iPSC-derived cardiomyocytes. Conclusions: Cardiomyocytes can be efficiently generated from human urine, through the cellular reprogramming technology. DMD cardiomyocytes retained the patient’s genetic information and manifested a dystrophin-null phenotype. Functional assessments are underway to determine differences that may exist between genotypes.

Generation of Neural Cells from DM1 Induced Pluripotent Stem Cells As Cellular Model for the Study of Central Nervous System Neuropathogenesis

Dystrophia myotonica type 1 (DM1) is an autosomal dominant multisystem disorder. The pathogenesis of central nervous system (CNS) involvement is poorly understood. Disease-specific induced pluripotent stem cell (iPSC) lines would provide an alternative model. In this study, we generated two DM1 lines and a normal iPSC line from dermal fibroblasts by retroviral transduction of Yamanaka's four factors (hOct4, hSox2, hKlf4, and hc-Myc). Both DM1 and control iPSC clones showed typical human embryonic stem cell (hESC) growth patterns with a high nuclear-to-cytoplasm ratio. The iPSC colonies maintained the same growth pattern through subsequent passages. All iPSC lines expressed stem cell markers and differentiated into cells derived from three embryonic germ layers. All iPSC lines underwent normal neural differentiation. Intranuclear RNA foci, a hallmark of DM1, were detected in DM1 iPSCs, neural stem cells (NSCs), and terminally differentiated neurons and astrocytes. In conclusion, we have successfully established disease-specific human DM1 iPSC lines, NSCs, and neuronal lineages with pathognomonic intranuclear RNA foci, which offer an unlimited cell resource for CNS mechanistic studies and a translational platform for therapeutic development.

A Single Chronic Myeloid Leukemia (CML) Stem Cell Harboring Both BCR-ABL and JAK2-V617F Mutation Modeled by Induced Pluripotency

AbstractAbstract 3728The occurrence of JAK2-V617F mutation has been reported during the evolution of several patients with Ph1-positive CML. In all cases where a clonal analysis has been performed, the involvement of two different hematopoietic stem cells (HSC) has been demonstrated, with the presence of two different myeloproliferative disorders (MPDs). The occurrence of the V617F mutation in a leukemic stem cell (LSC) harboring the BCR-ABL rearrangement has not been reported so far. In a 63- year old patient with a diagnosis of high Sokal score Ph1+ CML, the initial therapy with Imatinib led to efficient cytoreduction and complete cytogenetic response but with persistent splenomegaly. Upon disease progression, he was treated successively with IFN-alpha, ARA-C, Dasatinib and Nilotinib with transient responses and persistent splenomegaly. The progressive increase of platelet counts led to the discovery of JAK2-V617F mutation, which appeared to be present from the diagnosis of CML with a progressive increase (40 % at diagnosis up to 70% at the time of the discovery of JAK2 mutation). To determine if BCR-ABL and JAK2 mutation were present in the same cells, a clonogenic assay was performed and single BFU-E’s (n=11) were analyzed individually by q-RT-PCR. All 11 BFU-E expressed BCR-ABL at high levels with a mean BCR-ABL/ABL ratio of 157% (Range 91–207 %). The same individual BFU-E also revealed the presence of a heterozygous JAK2-V617F mutation. To generate a HSC model harboring simultaneously these two major molecular events, we have generated induced pluripotent stem cells (iPSC) from leukemic cells using Sendai-virus mediated Oct4, Sox2, Klf4 and c-Myc gene transfer. We have also generated iPSC from non-leukemic (BCR-ABL-negative, JAK2-V617F-negative) endothelial progenitor cells (EPC) obtained from peripheral blood. iPSC generated from both sources expressed pluripotency markers (Tra-1–60, SSEA-3, SSEA-4) and generated teratoma in NOD/SCID mice. In 24 individual leukemic iPSC clones which were amplified, Ph1-chromosome was found to be present in all mitoses and BCR-ABL was highly expressed (Mean ratio 140%). Each iPSC exhibited the same V617F mutation which co-existed in the primary leukemic BFU-E. None of the non-leukemic iPSC expressed BCR-ABL neither V617F. To determine the hematopoietic potential of both iPSC, we have generated day-16 embryoid bodies (EB) followed by induction of hematopoiesis. Day 16 EB’s generated variable numbers of CD34+ cells (12–40 %, n=4) and clonogenic potential (25–340 CFC/105 D16-EB) with evidence of growth-factor-independent CFC’s. Interestingly, IM and Dasatinib had partial inhibitory effects on CFC activity, but a combination of Pimozide (STAT5 inhibitor) and IM had a major synergistic effect. As neither normal iPSC nor human embryonic stem cells (hESC) are able to generate definitive, long-term hematopoiesis, we have tested the long-term HSC potential of these iPSC in long-term culture initiating cell (LTC-IC) assays as compared to human ESC line H1. In these conditions, leukemic iPSC generated hematopoietic cells with terminal granulocytic and erythroblastic differentiation and gave rise for the first time to an LTC-IC- derived progeny at 6 weeks (200 CFU-C/105 Day-16 EB) whereas in the same time H1-derived cells did not generate any significant LTC-IC potential (3 CFU-C/105 D-16 EB). Interestingly, non-leukemic iPSC generated also hematopoietic cells but exhibited a major genetic instability in culture with appearance after several passages, of a t (5;7) translocation and major deletions/duplications in CGH arrays. To determine a potential upstream molecular event which might be involved at the origin of the occurrence of BCR-ABL and JAK2 mutation in the same HSC, a whole genome sequencing strategy is underway. In conclusion, we report for the first time the occurrence of two major molecular events involved in CML and non-CML MPDs in the same LSC and demonstrate the utility of iPSC modeling to study the earliest HSC involved. These results suggest strongly that the co-existence of BCR-ABL and V617F enables the specification of iPSC towards hematopoieis which could lead to the identification of specific hematopoietic signaling pathways. Finally, clonal analysis of hematopoietic cells in CML patients with demonstration of Jak2V617 mutations is worthwhile to determine the prevalence of the simultaneous occurrence of these two molecular events in the same cell. Disclosures:Turhan:Novartis, Bristol Myers Squibb: Honoraria, Research Funding.