iPS Cell Lines Research Articles

Derivation of human iPS cell lines from monozygotic twins in defined and xeno free conditions

Human induced pluripotent stem (hiPS) cell lines CTRL-9-II and CTRL-10-I were derived from healthy monozygotic twin donors using non-integrating RNA based Sendai virus reprogramming and cultured in a xeno-free chemically defined condition. The established hiPS cell lines, CTRL-9-II and CTRL-10-I, are karyotypically normal, free from reprogramming vectors, display endogenously expression of pluripotency factors at levels similar to embryonic stem cells. The generated iPS cell lines demonstrate pluripotency by passing bioinformatics assay PluriTest and by embryonic body assay.

Generation of a disease-specific iPS cell line derived from a patient with Charcot-Marie-Tooth type 2K lacking functional GDAP1 gene

Human CMT2-FiPS4F1 cell line was generated from fibroblasts of a patient with Charcot-Marie-Tooth disease harbouring the following mutations in the GDAP1 gene in heterozygosis: p.Q163X/p.T288NfsX3. This patient did not present mutations in the PM22, MPZ or GJB genes. Human reprogramming factors OCT3/4, KLF4, SOX2 and C-MYC were delivered using a non-integrative methodology that involves the use of Sendai virus.

The Effects of the Neutrophil Elastase Inhibitors MK0339 and Sivelestat on the Survival, Proliferation and Maturation of iPSC and HL60 Cells Expressing Mutant Neutrophil Elastase

Background: Mutations in ELANE, the gene for neutrophil elastase (NE), are the most frequent cause of both cyclic neutropenia (CyN) and severe congenital neutropenia (SCN). G-CSF and HSCT are currently the only effective treatment options. We have examined the possibility that inhibitors of NE might also be treatment options.Methods:Cells: HL60 human promyelocytic cells, a commercially available cell line, and induced pluripotent stem cells (iPSc) derived from patient dermal fibroblasts or bone marrow stromal cells, reprogrammed using episomal vectors.NE inhibitors: Two cell permeable inhibitors were used: MK0339 provided by Merck and sivelestat from Sigma.Results:Expression of mutant NE in HL60 CellsTransient Expression:Transient transfection of HL60 cells with 4 different ELANE mutants (P139L, C151Y, V174_C181del, G214R) cloned in pcDNA 3.1 vector showed increased apoptosis after 48h of culture in 10% FBS RPMI and expression of mutant constructs. Addition of Merck NE inhibitor MK0339 with 1uM final concentration in culture significantly improved survival of cells expressing each of the mutations tested detected by Annexin V staining using flow cytometry. (p<0.0001) The effect of the sivelestat (100nM) was significantly effective on only G214R mutant. (p= 0.035)Regulated Expression:We tested the effects of Merck inhibitor MK0339 on tet-inducible HL60 cells with regulated expression of mutant NE (V174_C181del), a mutation found in a patient with severe congenital neutropenia who died of AML. We examined 3 characteristics of these cells: cell survival, myeloid differentiation, and proliferation. The cell lines, expressing wild type or mutant NE under a tetracycline regulated promoter, were treated with MK0339 and cultured for 72 hours in the presence or absence of doxycycline. MK0339 significantly reduced mutant NE triggered apoptosis and increased granulocytic differentiation induced by addition of all-trans retinoic acid (ATRA) in 5-day cultures. Expression of mutant NE also significantly reduced the growth rate of the HL60 cells, but addition of MK0339 promoted growth of these HL60 cells.Induced Pluripotent Stem Cells (iPSC)We have established iPS cell lines from three neutropenic patients (two brothers with P139L and another patient with G214R) and two healthy volunteers. Cell lines from all 3 patients consistently grew much slower and formed much smaller colonies than the controls using both mTeSR1/matrigel and DEF-CS feeder free culture medium systems. We observed more than 3-fold growth reduction of patient derived lines compared to healthy volunteers. We therefore tested the effects of MK-0339 and sivelestat under the same culture conditions. Growth of patient cell lines was enhanced and the cells appeared more robust, especially after addition of 1uM MK0339. The latter normalized patient derived iPSc proliferation. We also investigated survival of the iPS cells during growth in feeder free settings and the effect of the NE inhibitors. After 4 days of culture in DEF-CS system, the patient derived cell lines showed increased apoptosis measured by Annexin V. We observed more than 9-fold difference in increased apoptosis of patient derived cells compared to healthy volunteers. Addition of MK-0339 and sivelestat reduced the apoptosis. MK0339 significantly reduced the apoptosis in both mutant cell lines, while sivelestat significantly reduced the apoptosis in only P139L mutant cell lines. Again, sivelestat appeared to be less effective.Myeloid differentiation of iPSc was also impaired and inefficient in patient derived cell lines detected with granulocytic differentiation markers using flow cytometry. Introduction of MK0339 in this system normalizes the impaired myeloid differentiation capacities of patient derived iPSc lines.Summary/Conclusion: These studies clearly show effects of these selected inhibitors to enhance neutrophil formation in reproducible cellular models that mimics features of SCN and CyN. We believe this work will lead to novel therapies for ELANE associated neutropenia and open the door to other new therapies for neutropenia. DisclosuresDale:Amgen: Consultancy, Honoraria, Research Funding.

Multifactorial Optimizations for Directing Endothelial Fate from Stem Cells.

Embryonic stem cells (ESC) and induced pluripotent stem (iPS) cells are attractive in vitro models of vascular development, therapeutic angiogenesis, and tissue engineering. However, distinct ESC and iPS cell lines respond differentially to the same microenvironmental factors. Developing improved/optimized differentiation methodologies tailored/applicable in a number of distinct iPS and ESC lines remains a challenge in the field. Currently published methods for deriving endothelial cells (EC) robustly generate high numbers of endothlelial progenitor cells (EPC) within a week, but their maturation to definitive EC is much more difficult, taking up to 2 months and requiring additional purification. Therefore, we set out to examine combinations/levels of putative EC induction factors—utilizing our stage-specific chemically-defined derivation methodology in 4 ESC lines including: kinetics, cell seeding density, matrix signaling, as well as medium treatment with vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF). The results indicate that temporal development in both early and late stages is the most significant factor generating the desired cells. The generation of early Flk-1+/KDR+ vascular progenitor cells (VPC) from pluripotent ESC is directed predominantly by high cell seeding density and matrix signaling from fibronectin, while VEGF supplementation was NOT statistically significant in more than one cell line, especially with fibronectin matrix which sequesters autocrine VEGF production by the differentiating stem cells. Although some groups have shown that the GSK3-kinase inhibitor (CHIR) can facilitate EPC fate, it hindered the generation of KDR+ cells in our preoptimized medium formulations. The methods summarized here significantly increased the production of mature vascular endothelial (VE)-cadherin+ EC, with up to 93% and 57% purity from mouse and human ESC, respectively, before VE-cadherin+ EC purification.

IPS-derived neural progenitor cells from PPMS patients reveal defect in myelin injury response

Primary progressive multiple sclerosis (PPMS) is a chronic demyelinating disease of the central nervous system (CNS) currently lacking any effective treatment. Promoting endogenous brain repair offers a potential strategy to halt and possibly restore neurologic function in PPMS. To understand how the microenvironment within white matter lesions plays a role in repair we have focused on neural progenitor cells (NPCs) since these are found in lesions in PPMS and have been found to influence oligodendrocyte progenitor cell maturation (OPCs). To better understand the cellular nature of NPCs in PPMS we developed iPS cells from blood samples of PPMS patients and age matched non-disease spouse or blood relative controls. Using these iPS cell lines we determined that the NPCs from PPMS cases provided no neuroprotection against active CNS demyelination compared to NPCs from control iPS lines which were capable of completely preventing injury. Conditioned media (CM) from PPMS NPCs provides no protection to OPCs and prevents maturation of OPCs into oligodendrocytes in vitro. We also found that CM from PPMS iPS NPCs elicited patient-specific differences in the response to compounds that should foster oligodendrocyte (OL) maturation. Together, these data establish a new model for understanding the nature of myelination defects in PPMS which may lead to novel targeted approaches for preventing demyelination in these patients.

Efficient Generation of β-Globin-Expressing Erythroid Cells Using Stromal Cell-Derived Induced Pluripotent Stem Cells from Patients with Sickle Cell Disease

Human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells represent an ideal source for in vitro modeling of erythropoiesis and a potential alternative source for red blood cell transfusions. However, iPS cell-derived erythroid cells predominantly produce ε- and γ-globin without β-globin production. We recently demonstrated that ES cell-derived sacs (ES sacs), known to express hemangioblast markers, allow for efficient erythroid cell generation with β-globin production. In this study, we generated several iPS cell lines derived from bone marrow stromal cells (MSCs) and peripheral blood erythroid progenitors (EPs) from sickle cell disease patients, and evaluated hematopoietic stem/progenitor cell (HSPC) generation after iPS sac induction as well as subsequent erythroid differentiation. MSC-derived iPS sacs yielded greater amounts of immature hematopoietic progenitors (VEGFR2 + GPA-), definitive HSPCs (CD34 + CD45+), and megakaryoerythroid progenitors (GPA + CD41a+), as compared to EP-derived iPS sacs. Erythroid differentiation from MSC-derived iPS sacs resulted in greater amounts of erythroid cells (GPA+) and higher β-globin (and βS-globin) expression, comparable to ES sac-derived cells. These data demonstrate that human MSC-derived iPS sacs allow for more efficient erythroid cell generation with higher β-globin production, likely due to heightened emergence of immature progenitors. Our findings should be important for iPS cell-derived erythroid cell generation. Stem Cells 2017;35:586-596.

Lymphoblast-derived integration-free iPS cell line from a female 67-year-old Alzheimer's disease patient with TREM2 (R47H) missense mutation

Human lymphoblast cells from a female patient diagnosed with Alzheimer's disease (AD) possessing the missense mutation TREM2 p.R47H were used to generate integration-free induced pluripotent stem cells (iPSCs) employing episomal plasmids expressing OCT4, SOX2, NANOG, LIN28, c-MYC and L-MYC. The iPSCs retained the TREM2 mutation, and were defined as pluripotent based on (i) expression of pluripotent-associated markers, (ii) embryoid body-based differentiation into cell types representative of the three germ layers and (iii) the similarity between the transcriptomes of the iPSC line and the human embryonic stem cell line H1 with a Pearson correlation of 0.961.

Generation of human iPS cell line CTL07-II from human fibroblasts, under defined and xeno-free conditions

CTL07-II is a healthy feeder-free and characterized human induced pluripotent stem (iPS) cell line. Cultured under xeno-free and defined conditions. The line is generated from healthy human fibroblasts with non-integrating Sendai virus vectors encoding the four Yamanaka factors, OCT4, SOX2, KLF4 and cMYC. The generated iPS cells are free from reprogramming vectors and their purity, karyotypic stability and pluripotent capacity is confirmed.

Murine transgenic iPS cell line for monitoring and selection of cardiomyocytes

We report here a transgenic murine induced pluripotent stem cell (iPSC) line expressing puromycin N-acetyltransferase (PAC) and enhanced green fluorescent protein (EGFP) under the control of α-myosin heavy chain promoter. This transgenic cell line reproducibly differentiates into EGFP-expressing cardiomyocytes (CMs) which can be generated at high purity with puromycin treatment and exhibit molecular and functional properties of immature heart muscle cells. This genetically modified iPSC line can be used for assessment of the utility of CMs for myocardial repair, pharmacological and toxicological applications and development of improved cardiac differentiation protocols.

Inhibition of the integrin signal constitutes a mouse iPS cell niche.

Stem cells are regulated by their surrounding microenvironments, called niche, such as cell-cell interaction and extracellular matrix. Classically, feeder cells as a niche have been used in the culture of iPS cells from both the mouse and the human. However, the regulation mechanism of stem cells by feeder cells as a niche still have been partially unclear. In this study, we used three murine iPS cell lines, iPS-MEF-Ng-20D-17, iPS-MEF-Ng-178B-5 and iPS-MEF-Fb/Ng-440A-3, which were generated by different reprogramming methods. In general, these cell lines commonly need the feeder cells as a niche to culture. Recently, the effect of substrate stiffness is known in stem cell study. First, we focused on the mechanical properties of feeder cells, and then we speculated that feeder-less culture might be made possible by using molecules in place of the mechanical properties of the niche. Finally, we found that the combination of disintegrin (echistatin) and 2i (GSK3 inhibitor and MEK inhibitor) is a sufficient condition for three murine iPS culture. This novel method of mimicking the murine iPS cell niche may be useful to understand signaling pathways to maintain the pluripotency of stem cells.

Neurons Derived From Patient-Specific Induced Pluripotent Stem Cells: a Promising Strategy Towards Developing Novel Pharmacotherapies for Autism Spectrum Disorders

Neurons Derived From Patient-Specific Induced Pluripotent Stem Cells: a Promising Strategy Towards Developing Novel Pharmacotherapies for Autism Spectrum Disorders

Generation of human iPS cell line SKiPSc1 from healthy Human Neonatal Foreskin Fibroblast cells

The SKiPSc1 induced pluripotent stem (iPS) cell line was generated from Human Neonatal Foreskin Fibroblasts (HNFFs) obtained from a healthy donor infant that were reprogrammed using non-integrating Sendai viral vectors expressing Oct3/4, Sox2, c-Myc, and Klf4.

Generation of urine iPS cell lines from patients with Attention Deficit Hyperactivity Disorder (ADHD) using a non-integrative method

Urine samples were collected from three patients (2 males, 1 female) with clinically diagnosed Attention Deficit Hyperactivity Disorder (ADHD) according to DSM-5 criteria using semi-structured interviews (K-SADS adapted for adults) by a trained psychiatrist. Urine epithelial cell lines were established and expanded for subsequent reprogramming procedure. Induced pluripotent stem cells (iPSCs) were derived using integration-free CytoTune®-iPS 2.0 Sendai Reprogramming Kit, which includes Sendai virus particles of the four Yamanaka factors Oct3/4, Sox2, Klf4 and c-Myc.

Generation of urine iPS cell line from a patient with obsessive-compulsive disorder using a non-integrative method

Urine sample was collected from a 29-year-old male patient with an early onset form of DSM-5 obsessive-compulsive disorder (OCD), comorbid body dysmorphic and excoriation (skin-picking) disorders, and a positive family history for OCD. Urine cell line was established and expanded for the reprogramming procedure. Induced pluripotent stem (iPS) cells were derived using the integration-free CytoTune®-iPS 2.0 Sendai Reprogramming Kit, which includes Sendai virus particles of the four Yamanaka factors Oct3/4, Sox2, Klf4 and c-Myc.

Generation of iPS cell lines from schizophrenia patients using a non-integrative method

Skin biopsies were collected from three male patients (age 45, 44 and 44) with clinically diagnosed schizophrenia. The patients were diagnosed according to DSM-5 criteria by a trained psychiatrist. Dermal fibroblast cell lines were established and expanded for subsequent reprogramming procedures. Induced pluripotent stem (iPS) cells were derived using the integration-free CytoTune®-iPS 2.0 Sendai Reprogramming Kit, containing Sendai virus particles of the four Yamanaka factors Oct3/4, Sox2, Klf4 and c-Myc.

312. Gene Correction of LGMD2A Patient-Specific iPS Cells for Targeted Autologous Cell Therapy

Limb girdle muscular dystrophy type 2A (LGMD2A), an incurable autosomal recessive disorder and the most common form of LGMD, is characterized by symmetric and progressive weakness of limb-girdle muscles leading to loss of ambulation. LGMD2A is caused by mutations in the Calpain 3 (CAPN3) gene which encodes a muscle-specific intracellular calcium-sensitive cysteine protease, hypothesized to act as a gate keeper for proper assembly and maintenance of the sarcomere. A cell therapy based on induced pluripotent stem (iPS) cells has the potential for treating muscular dystrophy. We have demonstrated that transplantation of skeletal muscle progenitors obtained from disease-free human iPS cells can restore muscle contractility in a mouse model for Duchenne Muscular Dystrophy. The goal of this study is to apply gene editing tools to correct the CAPN3 mutation in LGMD2A iPS cells, and to determine the feasibility of an iPS-cell based autologous cell therapy for LGMD2A. Here we investigated two approaches to genetically correct a LGMD2A patient-specific iPS cell line with homozygous deletion of exon 17 to 24 in the CAPN3 gene. The first approach consisted of knocking in the coding region of CAPN3 cDNA with SV40 polyadenylation signal sequence downstream in exon 1 of the endogenous CAPN3 gene by CRISPR-CAS9 induced double stranded break (DSB) mediated homology directed DNA repair from exogenous donor template, allowing for universal correction of all types of CAPN3 mutations. PCR, sequencing and Southern blot analysis on iPS colonies post- genome editing confirmed the integration of the cDNA in exon 1 of the CAPN3 gene. The second approach involved inserting the missing exons 17-24 with SV40 polyadenylation signal sequence downstream contiguous with the exon 16 of the endogenous CAPN3 gene. PCR and sequencing analysis on iPS colonies post-genome editing confirmed proper integration in this approach as well. LGMD2A iPS cell clones, genetically corrected using these two gene editing methods will be differentiated into skeletal muscle progenitors using conditional expression of Pax7, as previously described by our group, and evaluated in vitro and in vivo for restoration of full length CAPN3. For the latter, we have generated an immunodeficient LGMD2A mouse model by combining the CAPN3-KO mouse model with NOD/SCID and IL2Rg mutations. This mouse is ideal for studies on the transplantation of human cells. In summary, we have corrected the CAPN3 mutation in LGMD2A iPS cells for the first time, and ongoing transplantation studies with corrected iPS cells will provide proof-of-principal for a potential new cell therapy that can permanently restore CAPN3 function in all LGMD2A patients.

Human Brain Microvascular Endothelial Cells Derived from the BC1 iPS Cell Line Exhibit a Blood-Brain Barrier Phenotype.

The endothelial cells that form capillaries in the brain are highly specialized, with tight junctions that minimize paracellular transport and an array of broad-spectrum efflux pumps that make drug delivery to the brain extremely challenging. One of the major limitations in blood-brain barrier research and the development of drugs to treat central nervous system diseases is the lack of appropriate cell lines. Recent reports indicate that the derivation of human brain microvascular endothelial cells (hBMECs) from human induced pluripotent stem cells (iPSCs) may provide a solution to this problem. Here we demonstrate the derivation of hBMECs extended to two new human iPSC lines: BC1 and GFP-labeled BC1. These hBMECs highly express adherens and tight junction proteins VE-cadherin, ZO-1, occludin, and claudin-5. The addition of retinoic acid upregulates VE-cadherin expression, and results in a significant increase in transendothelial electrical resistance to physiological values. The permeabilities of tacrine, rhodamine 123, and Lucifer yellow are similar to values obtained for MDCK cells. The efflux ratio for rhodamine 123 across hBMECs is in the range 2–4 indicating polarization of efflux transporters. Using the rod assay to assess cell organization in small vessels and capillaries, we show that hBMECs resist elongation with decreasing diameter but show progressive axial alignment. The derivation of hBMECs with a blood-brain barrier phenotype from the BC1 cell line highlights that the protocol is robust. The expression of GFP in hBMECs derived from the BC1-GFP cell line provides an important new resource for BBB research.

Premature ovarian failure patient‐specific induced pluripotent stem (iPS) cells show compromised potential to develop to germ cells and granulosa cells

Premature ovarian failure (POF), also called primary ovarian insufficiency (POI), is a common endocrine disorder in females, accounting for 1–2% in women during the reproductive ages. Patients with the disease experience spontaneous menopause before the age of 40 years, and frequently encounter significant difficulties and pains in overcoming the problems of infertility and menopausal syndrome. However, the mechanisms underlying POF are difficult to study due to the lack of a suitable disease model. Recently pluripotent stem cells, including human embryonic stem (ES) cells and human induced pluripotent stem (iPS) cells, are becoming useful cell sources to study and dissect the molecular mechanisms underlying in vitro differentiation into specialized cell types. We and other researchers have previously shown that pluripotent stem cells including human ES cells and iPS cells are capable of developing into germ cells and granulosa cells. In this study, we derived POF patient‐specific iPS cell lines from three patients and two controls using skin fibroblasts or PBMCs and induced them to early female germ cells expressing germ cell markers VASA and GDF9 and meiosis marker SCP3, and also to granulosa‐like cells expressing FOXL2, FSHR, AMHR2, AMH and CYP19A1 (aromatase). We observed that the differentiated POF‐patient iPS cells showed differential expression levels of the granulosa cell markers and VASA, when compared to NTU1 human embryonic stem cells. The data demonstrated that iPS cells can be produced from POF patients and control subjects, and these cells can be induced in vitro to early germ cells and granulosa cells, but significantly with compromised potential when compared to other pluripotent stem cells. The POF patient‐specific iPS cells derived in this study thus represent a very useful disease model for further studies to test the disease mechanisms and to test drugs that may be useful for treating POF.Support or Funding InformationNational Science Council of the Republic of China (NSC) and National Taiwan University Hospital (NTUH), Taiwan

Donor Dependent Variations in Hematopoietic Differentiation among Embryonic and Induced Pluripotent Stem Cell Lines.

Hematopoiesis generated from human embryonic stem cells (ES) and induced pluripotent stem cells (iPS) are unprecedented resources for cell therapy. We compared hematopoietic differentiation potentials from ES and iPS cell lines originated from various donors and derived them using integrative and non-integrative vectors. Significant differences in differentiation toward hematopoietic lineage were observed among ES and iPS. The ability of engraftment of iPS or ES-derived cells in NOG mice varied among the lines with low levels of chimerism. iPS generated from ES cell-derived mesenchymal stem cells (MSC) reproduce a similar hematopoietic outcome compared to their parental ES cell line. We were not able to identify any specific hematopoietic transcription factors that allow to distinguish between good versus poor hematopoiesis in undifferentiated ES or iPS cell lines. There is a relatively unpredictable variation in hematopoietic differentiation between ES and iPS cell lines that could not be predicted based on phenotype or gene expression of the undifferentiated cells. These results demonstrate the influence of genetic background in variation of hematopoietic potential rather than the reprogramming process.

Abstract A08: Using directed differentiation of human pluripotent stem cells and gene expression profiling to characterize the cell of origin of neuroblastoma

Abstract Purpose: Neuroblastoma (NB) is one of the most common childhood tumors, accounting for ~15% of all pediatric cancer deaths. Improved neuroblastoma disease modeling is needed to improve our understanding and ultimately lead to the development of more effective therapies. NB is an embryonal tumor of the sympathetic nervous system believed to arise from sympathoadrenal (SA) precursor cells belonging to the primitive neural crest. Because the neural crest is a transient embryologic structure, there have been few studies comparing NB tumors to normal human neural crest stem cells (NCSC). However, recent advances have enabled the in vitro differentiation of neural crest cells from pluripotent stem cells. We compared gene expression profiles of pluripotent stem cells, NCSC, and NB tumors to characterize the gene expression profile of the putative cell of origin of NB as the first step in the development of a human NCSC based model of NB. Methods: Human embryonic and induced pluripotent stem (iPS) cells were differentiated to early NCSC and SA precursors in vitro using modifications of previously published protocols. Specifically, undifferentiated human embryonic stem cell GFP reporter and iPS cell lines were induced to differentiate into SA cells in a sequential, stepwise manner over the course of ~4 weeks. After differentiation, SA precursors were identified by expression of the lineage-specific transcription factors ASCL1 and PHOX2B, as well as other markers including tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH). We subsequently compared the gene expression profiles of these cells to early, multi-potent neural crest stem cells, autonomic and enteric neurons, 12 NB cell lines and 39 non-MYCN amplified primary NB tumors (3 stage 1, 5 stage 2, 3 stage 3, 24 stage 4, and 4 stage 4S) using Illumina HT-12 bead arrays. These profiles were also compared to the gene expression profiles of laser-capture microdissected fetal adrenal neuroblasts previously reported by De Preter et. al. (Genome Biology 2006, 7:R84). Results were normalized using quantile normalization and analyzed with principle components analysis (PCA); differential expression analysis was performed using the Linear Models for Microarray Data (Limma) method. Pathway enrichment analysis was performed using DAVID annotation tools and cross-platform normalization was performed using the CONOR package in the R software program. All analyses were corrected for multiple comparisons and results considered significant when False Discovery Rate (FDR) was &amp;lt;0.05. Results: PCA showed that undifferentiated iPS cells and early NCSC cluster relatively closely together and away from primary tumors and fetal adrenal neuroblasts. The NB cell lines and primary tumors do not cluster closely together suggesting there are significant differences between them. Gene expression profiles of in vitro differentiated ASCL1 and PHOX2B(+) SA precursor cells were similar to fetal adrenal neuroblasts and more closely resembled NB tumors than did earlier multi-potent NCSC. Comparison between SA precursor cells, NCSC, and primary tumors identified numerous differentially expressed genes (FDR&amp;lt;0.05) the majority of which are regulators of embryologic and/or neural development (SOX3, DPPA4, HES5, LIN28, IRX2, SOX21, PAX6, FABP7, and OTX2 among others). Most were overexpressed in normal neural crest derivatives relative to NB cell lines and tumors. Conclusions: This study, the first of its kind for this disease, demonstrates the feasibility and potential utility of using human pluripotent stem cells and in vitro derived sympathoadrenal precursors cells to simulate early NB pathogenesis. Gene expression profiling of NB identified down regulation of multiple developmentally important genes of normal neural crest differentiation. Further refinement of this in vitro neuroblastoma tumor model as well as investigations of its disrupted gene pathways is underway. Note: This abstract was not presented at the conference. Citation Format: Stephen S. Roberts, Yudelca Ogando, Irina Ostrovnaya, Faranak Fattahi, Irene Cheung, Nai-Kong V. Cheung, Lorenz Studer, Mark Tomishima. Using directed differentiation of human pluripotent stem cells and gene expression profiling to characterize the cell of origin of neuroblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr A08.