New Jersey Stem Cell Research Symposium
Exploring academic and corporate stem cell research in New Jersey
|Showing 1 through 5 of a total of 41 abstracts.|
|PDCD2 expression correlates with disease status in acute leukemia patients
Barboza1, Nora, Svetlana Minakhina2, Daniel J. Medina1, Binaifer Balsara1, Sonya Greenwood1, Lien Huzzy1, Arnold B. Rabson3, Ruth Steward2 and Dale G. Schaar1 Affiliations: 1University of Medicine & Dentistry of New Jersey, Robert Wood Johnson Medical School, The Cancer Institute of New Jersey, New Brunswick, NJ, USA; 2 Waksman Institute, Rutgers University, Piscataway, NJ, USA; 3 Child Health Institute of New Jersey, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, New Brunswick, NJ, USA.
Abstract: PDCD2 is an evolutionarily conserved eukaryotic protein with unknown function. The Drosophila PDCD2 ortholog Zfrp8 has recently been shown to be essential for the maintenance of hematopoietic stem cells. Zfrp8 mutants exhibit a developmental delay, lethality during larval and pupal stages and marked hyperplasia of the hematopoietic organ, the lymph gland. The lymph gland overgrowth results from increased proliferation of undifferentiated hemocytes throughout development and abnormal hemocyte differentiation. Based on the above observations we hypothesized the aberrant PDCD2 expression may be present in human hematologic malignancies. We report that PDCD2 is highly expressed in acute leukemia cells relative to normal hematopoietic cells. Prospective analysis of PDCD2 expression in acute leukemia patients undergoing treatment indicates near universal high PDCD2 expression at diagnosis that falls dramatically as patients respond to therapy suggesting PDCD2 expression correlates with disease status.
Acknowledgements: This work was funded by a research grant provided to D.G.S by The New Jersey Commission on Cancer Research.
|Large-scale manufacture of custom TAL effector nucleases for genomic editing
Blackston, Veronica1, Poderycki, Michael1, Yang, Jian-Ping1, Matthias, Arnold2, Bodemann, Antje2, Arenskotter, Matthias2, Deinert, Michaela2, Kapur, Vinita1, Notka, Frank2, Wagner, Ralf2, Peterson, Todd1, Chesnut, Jon1 1Synthetic Biology Research & Development, Life Technologies, Carlsbad, CA, USA, 2Geneart, Regensburg, Germany
Abstract: Specific DNA-binding transcription activator-like effector nucleases (TALENS) are powerful tools for genome engineering with broad applications in a variety of cell types, including stem cells. The TAL binding domain, from Xanthamonas sp., governs the simple code used to assemble the DNA-binding specificity. We have developed large scale manufacture capability to custom-design TAL nuclease Gateway expression vectors that provide gene editing at targeted loci. In association with GENEART, a TALE monomer library has been created to generate rapid assembly of custom TALE proteins. We are working to optimize the TALE nuclease tool by comparing its activity in a variety of single expression vectors and developing a cell-free QC assay to measure cleavage and repair of specific loci. The TALEN expression clones are being assessed in mammalian cell lines and cell based models. It is expected that TAL effector nucleases will be used in precise genome engineering technologies such as generating IPSC clones, stably modified human embryonic stem cell clones and broader areas of regenerative medicine, research in disease models and the production of therapeutics.
|Ms. Pin-Fang Chen
|Tissue-specific RBFOX protein binding and imprinted expression at the UBE3A locus
Chen, P.-F., Lalande, M., Chamberlain, S.J., Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, CT
Abstract: Scientists have successfully reprogrammed human somatic cells to embryonic stem cell-like induced pluripotent stem cells (iPSCs), which can be differentiated into virtually any tissue in the human body. The ability to create iPSCs and their derivatives gives access to a variety of live human cells and provides tools for understanding genetic disease mechanisms. We have generated an in vitro model of Angelman syndrome (AS), a neurogenetic disorder, by reprogramming dermal fibroblasts from an individual with AS due to a large deletion of chromosome 15q11-q13 into iPSCs. AS is caused by loss of function of the imprinted gene UBE3A. In normal iPSCs, UBE3A is expressed from both parental alleles. The paternal allele of UBE3A becomes silenced during the process of neural differentiation, leading to the expression of UBE3A exclusively from the maternal allele in mature iPSC-derived neurons. This silencing occurs concomitantly with the paternal expression of RNA transcripts downstream of the SNURF/SNRPN gene, including an antisense transcript of UBE3A (UBE3A-ATS). Using AS iPSCs, we can observe the expression of UBE3A-ATS and repression of UBE3A sense transcript from the paternal allele alone during neural differentiation. We hypothesize that splicing changes that occur during neural differentiation result in neuron-specific processing of the downstream transcripts, including UBE3A-ATS, and that the neuron-specific processing of these transcripts leads to the silencing of paternal UBE3A. Using RNA immunoprecipitation with antibodies against the RBFOX1 family of splicing factors, we observed changes in the abundance of RBFOX paralogue binding to the paternal allele of the chromosome 15q11-q13 imprinted region as well as in other RBFOX-binding clusters during neural differentiation of AS iPSCs. Our results suggest that the RBFOX1 family of splicing factors may contribute to establishing the neuron-specific UBE3A imprint and that each RBFOX paralogue may have a unique function during neural differentiation.
Acknowledgements: Connecticut State Stem Cell Research Fund Raymond and Beverly Sackler Foundation NICHD R01HD68730-01
|Rick I. Cohen
|Design and Implementation of a Novel PiggyBac Mediated Transposition of Pluripotency Inducing Factors
Cohen, Rick, Jason Huang, Anthony Parel, Travis Antes*, Nandita Sarkar*, and Hye-Sook Kwon*. Rutgers University, Piscataway NJ. *System Biosciences Inc, Mountain View, CA
Abstract: Methodologies to create Induced Pluripotent Stem Cells (IPSCs) often use gene modifying technologies, as seen with the widely used retroviral based protocols. Others, like Lentiviral vectors have included CRE/Lox sites allowing near reversal of genetic modifications. While other non-gene-modifying methods, like episomal plasmids offer some advantages, often these are not as robust and reproducible as gene modifying technology. Here we have designed and implemented PiggyBac based transposon system with two choices of genes which include Oct4, Sox2, KLF4 and L-Myc or v-Myc/SV40-T together with an RFP or GFP tracer. Using standard techniques, we have modified human foreskin fibroblasts and followed optimized culture protocol for viral systems. While both systems allow for expression of the fluorescent marker, the v-Myc system was more robust and led to reproducible formation of putative IPSC colonies. Current work is underway to optimize simple gene transfer of these plasmids and to increase the efficiency of the L-Myc vector system. Our goals are to create a simple reversible system to generate IPSCs that would be robust and in the end, have no genetic modifications.
Acknowledgements: Supported in part by the Satell Foundation
|Mrs. Stephani A. Davis
|PDCD2 controls hematopoietic stem cell differentiation during development
Davis, S., Kramer, J., Granier, C.J., Piso, K., Rabson, A.B., and Sabaawy, H.E. Departments of Cellular & Molecular Pharmacology, Pathology and Laboratory Medicine, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Child Health Institute of New Jersey, and Cancer Institute of New Jersey, New Brunswick, NJ.
Abstract: Programmed cell death 2 (Pdcd2) is a highly conserved protein essential for maintenance of hematopoietic stem cells in Drosophila. The zebrafish has emerged as an ideal model to study hematopoiesis because zebrafish, similar to mammals, generate similar blood cell types from common hematopoietic stem cell lineages and the molecular signaling pathways involved are conserved. To investigate the role of Pdcd2 in hematopoietic lineage specification, we used morpholinos to knockdown Pdcd2 expression in zebrafish embryos. Pdcd2 knockdown led to an increase in progenitor and stem cell markers and caused accumulation of erythroid progenitors at the proerythroblast stage, indicating impaired hematopoietic development. Furthermore, hematopoietic stem cells (HSCs) capable of self-renewing and generating all blood lineages failed to emerge in the aorta-gonad mesonephros (AGM). Embryos with Pdcd2 knockdown also displayed mitotic defects with increased levels of p53-independent apoptosis. These hematopoietic defects were partially rescued by inhibiting Jak/Stat signaling and with the pdcd2 mRNA. These findings suggest a critical role for Pdcd2 in hematopoietic cell differentiation and lineage commitment.
Acknowledgements: We thank Neil Campbell for technical assistance, Leonard Zon for the Casper zebrafish, Robert Handin for the CD41-EGFP transgenic zebrafish, Raman Sood for multiple ISH probes, Jesus Torres-Vazquez for the ephrin probe. This work was supported by funds from the Cancer Institute of New Jersey.
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