NJ Stem Cell Technology Symposium

2009 NJ Stem Cell Technology Symposium

Jointly sponsored by Applied Biosystems, Inc. and the Rutgers Stem Cell Research Center. Held at the Bridgewater Marriott on Thursday, September 24, 2009. For more information, click here.

Scientific Session

Time	Presentation	Abstract (View/Hide All Abstracts)
8:00AM	Registration and poster set-up
8:45AM	Opening Address – Michael J. Pazzani, PhD - Rutgers University
9:00AM	BacMam Platform technologies for stem cells Uma Lakshmipathy	View/Hide Abstract
9:30AM	Immunosuppression Induced by Inflammatory Cytokines in Mesenchymal Stem Cells Guangwen Ren	View/Hide Abstract
10:00AM	Defining Stem Cell Niches to Direct Differentiation towards Neuroectoderm Lineages David I. Shreiber	View/Hide Abstract
11:00AM	Isolation of a Novel Rat Neural Precursor Clone that Efficiently Gives Rise to Functional GABAergic Neurons in Culture Martin Grumet	View/Hide Abstract
11:30AM	Single Cell Expression Analysis of the Embryonic Stem Cell to Neural Stem Cell Differentiation Process Richard Fekete	View/Hide Abstract
12:00PM	Molecular Genetic Characterization and Directed Differentiation of Trisomy 21 Induced Pluripotent Stem (iPS) Cells Nathan R. Treff	View/Hide Abstract
1:30PM	High Content Assay for the Identification of Small Molecule Modulators of hES Cells. Hakim Djaballah	View/Hide Abstract
2:15PM	DETECTION AND QUANTIFICATION OF STEM CELL GENE AND PROTEIN EXPRESSION USING A NOVEL INTEGRATED PCR APPROACH Shiaw-Min Chen	View/Hide Abstract
3:15PM	New microRNAs in human embryonic stem cells: Ago2 immunoprecipitation identifies RISC-associated RNAs that are regulated over differentiation Ronald P. Hart	View/Hide Abstract
3:45PM	Erythrocytic Differentiation of Placental Progenitor/Stem Cells Xiaokui Zhang	View/Hide Abstract

Technology Session

Time	Presentation
8:00AM–8:45AM	Registration and poster set-up
9:00AM–9:30AM	Next Generation siRNA Technology: Improved Efficacy, Potency, and Specificity for Cleaner RNAi Results – Rachele Kardon, Applied Biosystems
9:30AM-10:00AM	Real Time PCR Considerations: Do you know everything you need to succeed? - Mary Baltazar –Applied Biosystems
10:00AM–10:30AM	Quantitating microRNAs from small and precious samples Marcia Slater – Applied Biosystems
10:30AM-11:00AM	Poster Session
11:00AM–11:30AM	Jump-In Technology for the creation of engineered human embryonic stem cell lines -Uma Lakshmipathy, PhD – Invitrogen
11:30AM–12:00PM	A Novel Gene Expression Platform for Generating Inducible Pluripotent Stem Cells – Rick Cohen, PhD – Rutgers University
12:00PM–12:30PM	Culture and Characterization Systems for hESC, iPSC and hMSC - Lucas Chase, PhD –Invitrogen
12:30PM–1:15PM	Lunch & Poster Session
	Scientific Session only for Keynote Address
2:45PM–3:15PM	Poster Session
3:15PM – 3:45PM	New Protein Expression Assays using qPCR for the Detection and Relative Quantification of Protein Markers in Stem Cells Elana Swartzman, PhD – Applied Biosystems
3:45PM-4:15PM	SOLiD System 3: Introduction to Applied Biosystems Next Generation Sequencing System and its Applications to Your Research Jennifer Stover –Applied Biosystems

Posters

Poster number: 11
Bansal1, Nitu, Tulin Budak-Alpdogan1, Tom Davis2, Robert DiPaola1, Joseph R. Bertino1 1.Cancer Institute of New Jersey, NJ; 2.PTC Therapeutics, South Plainfield, NJ
Targeting prostate cancer stem like cells by novel Bmi-1 inhibitors View/Hide Abstract
Prostate cancer is the most commonly diagnosed male cancer in the developed world. Despite improved surgical and irradiation techniques, tumor relapse after therapy is commonly observed. Several studies have favored the existence of Cancer Stem cells (CSC) or tumor initiating cells in the tumor population. CSCs are defined as a population of cells found within a tumor that have characteristics similar to normal stem cells. Therefore targeting these CSCs could be an approach for effective prostate cancer treatment. Moreover, CSCs derived from advanced prostate tumors are hormone-independent and resistant to standard hormonal ablation therapy since they lack the androgen receptor; therefore, the rationale for developing prostate CSC-targeted drugs that can be used in combination with hormone therapy to accomplish total tumor eradication is substantial. In our studies we have identified a small population of tumor initiating cells from Du145, a prostate cancer cell line. Phenotype of this small subpopulation of CSCs is CD44hi/α2β1hi. These cells have higher migration, invasion and colony forming efficiency. Also, 1000 CD44hi/α2β1hi cells when injected in nude mice can form tumors. The Preliminary data also indicates that CD44hi/ α2β1hi cells have increased Bmi-1 levels. Bmi-1 (B-cell specific Moloney murine leukemia virus insertion site) is a member of the polycomb family of transcription factors responsible for the maintenance of self renewal in stem cells. In this study we demonstrate that novel inhibitor of Bmi-1, PTC-209, 210 and 211 (developed by PTC therapeutics) can inhibit Bmi-1 in Du145 cells and can also decrease the number of collagen attached cells therefore suggesting these can be used as a novel drug to target prostate cancer stem cells. These results encourage us to use this methodology to isolate cancer stem cells from human prostate cancer tissues and therefore will provide us with an opportunity to determine the effect of Bmi-1 inhibitors on human cancer stem cells.

Poster number: 12
Beauchamp1, Lesslie, Lingamanaidu Ravichandran1, Zhaohui Dong2, Laura Vozza-Brown2, Kristin Wiederholt2, and Susan Magdaleno1; 1Life Technologies, Austin, TX 78744; 2 Life Technologies, Carlsbad, CA 92008
Efficient Delivery of Highly Functional siRNAs using the Neon Transfection System to Characterize Cell Growth in Difficult-To-Transfect Human T-Lymphocyte Leukemia Cells View/Hide Abstract
siRNA mediated gene knockdown is a powerful experimental tool for reducing the expression of specific genes and is routinely used to identify gene function and elucidate biological pathways. Successful siRNA experiments require efficient delivery of highly functional and specific siRNAs into cells for guided cleavage of the target mRNA. While lipid-based transfection is a common approach for siRNA delivery, many cell types, including suspension cell lines and stem cells are not compatible with this technology. This limitation prevents the analysis of relevant gene targets in biologically appropriate cell types. The combination of highly functional Silencer Select siRNA reagents with the Neon Transfection System overcomes the limitations associated with lipid reagent transfection methods. Here we demonstrate the use of Ambion and Invitrogen technologies applied to hard to transfect Jurkat leukemia cancer cells to deliver siRNAs targeting specific genes involved in the G2/M checkpoint of the cell cycle and examine the effects on cell proliferation. First using the Neon Transfection System, we determined the best electroporation conditions to deliver siRNA into Jurkat. Three pre-set programs each with pulse voltage (1200, 1400, or 1600), width (20 or 30 msec), and one pulse number resulted in 90% or more mRNA knockdown and high cell viability. A dose response analysis revealed the most efficacious siRNA concentration to range from 15-50 pmol. Neon electroporation technology was found to be compatible with different siRNA designs. Using the optimized conditions, siRNA inhibition of three key proteins critical in mitotic progression, KIF11 (Eg5), WEE-1, and PLK-1, was analyzed to assess mRNA levels and cell proliferation in Jurkat. Results show that 15 pmol of siRNA is sufficient to silence each mRNA target ~ 80% or greater and demonstrate a significant loss in cell viability. The combination of highly functional siRNA reagents and the Neon Transfection System technology provides a robust tool for delivering siRNAs into cells that are difficult to transfect by lipid based methods and enables the use of RNAi strategies to identify and validate those genes important in diseases and biological processes like stem cell differentiation.

Poster number: 13
Camarillo, C., E. Oni, V. Fang, M. Swerdel, J.C. Moore, and R. P. Hart Rutgers Stem Cell Research Center, Rutgers University, Piscataway, NJ
Functional assay of new microRNAs in human embryonic stem cells View/Hide Abstract

Poster number: 14
Carlson, A. L.[1], Florek, C. A.[1], Kim, J. J.[1], Neubauer, T.[1], Moore, J.[2], Cohen, R.[2], Grumet, M.[2], Kohn, J.[3], Moghe, P. V.[1,4] [1] Department of Biomedical Engineering, Rutgers University, Piscataway, NJ 08854. [2] Department of Cell Biology and Neuroscience, Rutgers University, Piscataway, NJ 08854. [3] Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, NJ 08854. [4] Department of Chemical and Biochemical Engineering, Rutgers University, Piscataway, NJ 08854.
3-D Electrospun Polymer Scaffolds Promote Human Embryonic Stem Cell Self-Renewal and Controlled Organization View/Hide Abstract

Poster number: 15
Chapman1, Laura, Chad MacArthur2, Richard Fekete1, Ying Liu2 1 Life Technologies, Austin, TX; 2 Life Technologies, Carlsbad, CA
Pluripotency Gene Signature of Engineered BG01V/EG Stem Cells View/Hide Abstract
Life Technologies offers a complete workflow for profiling the gene expression of stem cells all the way from the actual cells and culturing reagents to the final profiles using TaqMan Assays. The hESC BG01v and STEMPRO® BG01V/EG are stem cell variants of the BG01 embryonic stem cell line and are available for purchase from Invitrogen. The BG01V/EG cells are reporter cells that have been engineered to express Emerald Green Fluorescent Protein (EmGFP) and the hESC BG01v contains a chromosomal target for site-specific insertion. The TaqMan® Gene Expression Cells-to-CT™ kit enables researchers to easily perform expression analysis using cultured cells without the purification of RNA. The kit includes reagents for cell lysis and RNA preservation, genomic DNA removal, reverse transcription (RT), and TaqMan Gene Expression Master Mix for real-time PCR analysis. The TaqMan® Array is a micro fluidic technology which uses 8 sample-loading ports, each connected to 48 reaction wells and run on the Applied Biosystems 7900HT Fast Real-Time PCR System. The Human Stem Cell Pluripotency Array contains a well-defined set of validated gene expression markers to characterize embryonic stem (ES) cell identity and assess phenotypic variations between embryonic stem cell isolates. In this study, hESC BG01v and STEMPRO® BF01V/EG cells were cultured to maintain stem cell morphology. An aliquot of 10,000 cells was prepared from each cell line using the TaqMan® Gene Expression Cells-to-CT™ kit. Resulting lysates were reverse transcribed, the cDNA loaded on the TaqMan Arrays and run on the 7900. The resulting data was normalized by subtracting the CT value of Beta Actin from all the other genes on the TaqMan array. These delta CTs were then compared to delta CTs of BG01 stem cells obtained in the same manner in a previously published paper (Nature Biotechnology, vol. 25, number 7). The two stem cell lines Life Tech offers closely matches those of the undifferentiated BG01 cells. They also have many differences in gene expression to the differentiated BG01 cells. This indicates that the even though the stem cells Life Technologies offers have been engineered and modified, they still maintain a similar gene expression profile of their parental line that has not been modified

Poster number: 16
Chopra, Anant, Pimchanok Pimton, Valerie Tutwiler, Peter Lelkes School of Biomedical Engineering, Drexel University
The Effect of Substrate Stiffness on the Differentiation of Murine Embryonic Stem Cells Towards Endoderm Lineage. View/Hide Abstract

Poster number: 17
Davila, J., L.A. Goff, C. Camarillo, M.R. Swerdel, J. C. Moore, R.I. Cohen, and R.P. Hart Rutgers Stem Cell Research Center, Rutgers University, Piscataway, NJ and The Broad Institute/CSAIL, MIT, Cambridge MA
Identification of Biologically Functional microRNAs in Human ESCs by Ago2 Immunoprecipitation and Sequencing View/Hide Abstract

Poster number: 18
Fecenko-Tacka, Karen, Shannon Morgan, Randall Allen, Kimberly Panckeri and Margaret A. Keller Coriell Institute for Medical Research, Camden, NJ 08103
A Biobank for Induced Pluripotent Stem Cells at Coriell Institute for Medical Research View/Hide Abstract
The Stem Cell Biobank, as part of Coriell Cell Repositories at Coriell Institute banks human and mouse stem cells for distribution for research purposes. The Stem Cell Biobank has been isolating, characterizing and distributing mesenchymal stem cells from cord blood and fat as well as hematopoietic progenitors for several years. These multipotent stem cells are useful for studies of lineage commitment and tissue engineering. With the advent of induced pluripotent stem (iPS) cells (see review Hockedlinger and Plath, Development 136:509, 2009) came a demand for a mechanism to bank and distribute this important stem cell to the research community. Further, proof of pluripotency of newly established iPS cell lines currently involves extensive molecular and cellular characterization that is both labor intensive and expensive for individual research labs. There is a need for not only a public bank of these critical new biomaterials, but also a streamlined assessment of pluripotency that would greatly simplify this process. Initially iPS cell lines were established using viral transformation of fibroblasts, more recently residue-free methods have been developed and other somatic cell types have been used as starting material. Many of the iPS lines described in the literature were derived from fibroblasts banked in the NIGMS Human Genetic Cell Repository and NIA Aging Cell Repository at Coriell. Induced pluripotent stem cell lines have been established from normal fibroblasts as well as fibroblasts from individuals with both Mendelian and complex diseases (Park et al, Cell 134:877, 2008). Disease-specific iPS cells are an invaluable tool with which researchers can examine the development of disease tissues in vitro. While the technology of cellular reprogramming continues to develop, it is crucial that the larger research community have access to these cell lines for basic research. During the past several months, the Stem Cell Biobank has established standard operating procedures (SOPs) for culturing, characterizing and banking pluripotent stem cells. Characterization of pluripotent stem cells includes both molecular and functional assays. Protocols for embryoid body formation and directed differentiation toward lineages representative of the three germ layers have been established. Surface expression and mRNA expression of pluripotency markers are also performed. In addition, quality control testing involves karyotype analysis, DNA fingerprinting and pathogen screening. A federally-approved human embryonic stem cell line, H9, was used to optimize all newly established procedures. These processes, when applied to submissions of iPS cell lines to the Coriell Cell Repositories will aid in determination of the best practices of iPS cell banking. Coriell is actively seeking submissions of existing iPS cell lines into its repository for purposes of distribution. Further, the Stem Cell Biobank at Coriell is now equipped to assist the individual researcher in characterizing their stem cell lines.

Poster number: 19
Ford, NR and Fresco, JR Princeton University, Princeton, NJ
Correction of the Sickle Cell Anemia Mutation in vivo View/Hide Abstract

Poster number: 20
Gokhale1, Paul, Janice Au-Young2,David Keys2, Srividya Dadi2, Shamit Soneji3, Mark Jones1, Neil Harrison1, Jon Sherlock2 ,Tariq Enver3, and Peter Andrews1 1: Centre for Stem Cell Biology, University of Sheffield, Sheffield. UK. 2: Applied Biosystems, Foster City, CA. USA. 3: The Weatherall Institute of Molecular Medicine, University of Oxford, Oxford. UK.
Quantitative single cell analysis of gene expression reveals lineage primed subsets within normal but not adapted self-renewing human ES cells View/Hide Abstract
Cell surface markers such as TRA-1-60 allow the identification and isolation of populations of undifferentiated ES cells free from their differentiated derivatives. Nevertheless, the resulting purified ES cells are heterogeneous which is likely to have implications for the directions that the cells, en masse, take during differentiation. Previously we showed that in human ES cultures the stem cell compartment can divided into several subsets based on expression of the surface antigen marker, SSEA3. We also noted that culture adaptation of human ES cells, which may involve marked karyotypic changes, may be accompanied by changes in the dynamics of the SSEA3(+) and (-) subsets. To examine the heterogeneity of these subsets further, we have now used TaqMan® assays to carry out a quantitative RT. PCR (Q.PCR) analysis of the expression of 96 genes in single cells isolated from the SSEA3(+) and (-) subsets of a normal and culture adapted sub-line of the human ES cell line, H7. Normal or culture adapted hES cells were electrostatically sorted, based on SSEA3 expression, as single cells into individual wells using flow cytometry. The resulting single cell samples were converted to cDNA and the cDNA pre-amplified using a pool of primers specific to the target genes to be interrogated. Amplified cDNA was subjected to TaqMan based Q.PCR in multi-well formats (TaqMan® Array micro fluidic card or 384-well plates). A global analysis of the data confirmed our previous research indicating that, as human embryonic stem cells adapt to culture, markers such as SSEA3 lose their ability to distinguish the most undifferentiated cells within a culture. However a more detailed examination revealed considerable heterogeneity of gene expression patterns in each of the subsets of cells. The extent of this heterogeneity is only apparent in these single cell analyses and would be obscured in population based analyses. Our data suggests of lineage priming and pre-patterning of undifferentiated cells, prior to commitment to differentiate, but that this effect is lost as a result of culture adaptation.
This work is supported by MRC, BBSRC, Yorkshire Cancer Research and the European Union (contract LSHG-CT-2006-018739 ,ESTOOLS)

Poster number: 21
Goldberg, Stanley I., Director, GLEN MILLS INC., Clifton, NJ 07014
BIOREACTOR DEVELOPED FOR LARGE-SCALE HIGH-DENSITY ADHERENT CELL GROWTH OVER EXTREMELY LONG CULTIVATION PERIODS (Z®RP) View/Hide Abstract

Poster number: 22
Karudapuram, S., Brewer M.T., Petrauskene, O., Sergeev, N., Bit, S., Brevnov, M., Brzoska, P., Liew SN., Liu JK and Furtado, M. Applied Biosystems, 850 Lincoln Center Drive, Foster City CA
Design, Development and Applications of the MycoSEQTM Myco Scan Mycoplasma Detection Assay View/Hide Abstract

Poster number: 23
Lieu1, Pauline, Ying Liu1, Steve Spusta2, Anette Kullmann3, Xianmin Zeng2, Berit Reed3, John Chesnut1, Karoline W. Schjetne3 1 Invitrogen Corporate, Life Technologies, Carlsbad, California, US 2 Buck Institute of Age Research, Novato, California, US 3 Invitrogen Dynal, Life Technologies, Oslo, Norway
DEPLETION OF SSEA-4+ HUMAN EMBRYONAL STEM CELLS USING DYNABEADS® View/Hide Abstract

Poster number: 24
Liu, Er, Hira Rizvi, Devang Patel,Department of Biomedical Engineering, Rutgers University Matt Becker, College of Polymer Science and Polymer Engineering, University of Akron Joachim Kohn, Ner Jersey Center for Biomaterials Prabhas Moghe, Department of Biomedical Engineering, Chemical and Biochemical Engineering, Rutgers University
Identification of stem cell phenotypes using a high content image cytometry based profiling methodology View/Hide Abstract
We developed a high-content image cytometry-based profiling methodology to investligate stem cell phenotypes, including normal state with stemness, lineage committed/differentiated state, abnormal/transformed state under various microenvironments within time frames much shorter than needed for traditional assays. This method generates a rich pool of parametrics of cell nuclei, namely “nuclear descriptors”, reporting nuclear morphology, chromatin associated proteins/nuclear protein expression, texture, structure and organization that can be identified as subcellular signatures of stem cells, to be used as indicator of cell cycle progression related phenotypes, e.g. proliferation/apoptosis, holding stemness, abnormal transformation, through subsequent exploratory data analysis including feature dimension reduction and classifications. To demonstrate its utility, we applied this methodology to study human mesenchymal stem cell (hMSC) differentiation and transformation under various soluble cues and substrate properties. The differentiated osteogenic and adipogenic cell phenotypes was evaluated through conventional functional assays, while hMSC transformation was evaluated through fluorescent in-situ hybridization (FISH) of a telomerase mRNA probe. Early profiling cell nuclear structure and organization using a nucleus matrix protein, nuclear mitotic apparatus (NuMA) demonstrated distinguishable early subcellular “signatures” when subjected to various differentiation cues or transformation. The methodology allowed for the information-rich screening of substrate mediated cellular behaviors, as well as the identification of nuclear features that seem to promote/inhibit abnormal cellular transformation or differentiation. This is a versatile technique that is amendable to the study of various stem cell type cultured under various conditions. It also has the potential to be high-throughput given the appropriate substrate platform.
RESBIO, supported by funding from the National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health

Poster number: 25
Liu, Jie, Nazia Khan, Elaine Han, Alan M. Graham and Shaohua Li Division of Vascular Surgery, Department of Surgery, UMDNJ-Robert Wood Johnson Medical School, New Brunswick, NJ 08903
iPS cells are less efficient than ES cells in germ layer formation in culture View/Hide Abstract

Poster number: 26
Liu, W.*, Bergenstock M.*, Lau, W.*, Sun, W.+, Liu, Q.* *3D Biotek, LLC, 675 US Highway 1, North Brunswick, NJ 08902 +Department of Mechanical Engineering, Drexel University, Philadelphia, PA 19104
Novel Poly-ε-caprolactone (PCL) Scaffolds For Three-Dimensional Stem Cell Differentiation View/Hide Abstract

Poster number: 27
Marchioni, M.A., Z. R. Mrowiec, G. Moskowitz, and D. Todd; Stem Cell Division, Community Blood Services, Allendale, New Jersey
Comparison of Umbilical Cord Blood TNC/Volume by Broad Race Groups View/Hide Abstract
BACKGROUND: Total nucleated cell count (TNC) is an important parameter used by laboratories and transplant centers to assess the adequacy of an umbilical cord blood (UCB) unit. TNC values may determine whether a unit is processed in the laboratory and whether the unit is deemed suitable for transplant. This study was undertaken to determine the effects, if any, of race on TNC of UCB units donated to a single public cord blood bank. STUDY DESIGN AND METHODS: 149 consecutive samples from UCB units donated to a public cord blood bank which contained data for race and volume between 50 and 120 ml were retrospectively analyzed for each of the following races: White (non-Hispanic), Black/African American, Mixed Race, White (Hispanic), and Asian. TNC was performed using the Sysmex XE-2100L analyzer on fresh umbilical cord blood samples. TNC was then divided by the volume of umbilical cord blood collected for each sample to eliminate the effect of collected volume on TNC. Data were grouped based on the race specified by the donor in the documentation accompanying the donation. The mean and standard deviation were calculated for each race. An un-paired t-test was performed comparing White (Non-Hispanic) data with the results of each of the other racial categories. Statistical significance for the p-test was set at p less than 0.05. RESULTS: Mean TNC counts per ml obtained were White (non-Hispanic) 17.21; Black/ African American 14.74; Mixed Race 16.13; White (Hispanic) 15.95; and Asian 15.49. Compared to mean TNC count per mL of units from White (non-Hispanic) donors, those from Black/African American donors (p<0.0001), Asian donors (p=0.0014), and White (Hispanic) donors (p=0.0281) contained significantly fewer TNC. Samples from UCB units from donors who self-identified as Mixed Race had lower TNC counts per mL, but the difference was not statistically significant (p=0.0518) when compared to those from White (non-Hispanic) donors. CONCLUSION: TNC counts of UCB units from Black/African American, Asian, and White (Hispanic) donors were significantly lower than those from White (non-Hispanic) donors. These results should be considered when determining the adequacy of UCB units; perhaps alternate TNC levels should be established for units from donors of different races.

Poster number: 28
Markov V, Modarai S, Gupta A, Guglielmi M, Parrillo J, Hollenberg S, and Saitta B. Coriell Institute for Medical Research, Camden, New Jersey, Rutgers University, Camden, New Jersey, Division of Cardiovascular Diseases, Cooper University Hospital, Camden, New Jersey.
Human Cord Blood Mesenchymal Stem Cells Respond to Hypoxic Cardiac Cell Injury in Vitro Resulting in Increased PDGF-AA, EGF and bFGF Secretion and Enhanced Cell Viability View/Hide Abstract

Poster number: 29
Moore, Jennifer C., Alana Toro-Ramos, Cindy Camarillo, Loyal A. Goff, Mavis Swerdel, Ronald P. Hart, and Rick I. Cohen Rutgers Stem Cell Research Center, Rutgers University, Piscataway, NJ
The Cell Cycle in Human Embryonic Stem Cells - Growth vs. Differentiation View/Hide Abstract

Poster number: 30
Mrowiec, Z. R. and D. Todd; Stem Cell Division, Community Blood Services, Allendale, New Jersey
Comparison of Controlled Versus Non-Controlled Rate Freezing of Umbilical Cord Blood Units View/Hide Abstract
BACKGROUND: Controlled rate freezing is recognized as a preferred method for cryopreservation of blood cells. DMSO, the cryoprotectant most commonly used, is well known to affect cell viability; thus, blood is typically frozen soon after the addition of DMSO. For umbilical cord blood (UCB), the addition of cryoprotectant may take between 5 and 10 minutes per unit. With large-scale processing, delays will occur, which could adversely affect cell viability when controlled rate freezing is utilized. This study was designed to compare the effect of controlled and non-controlled rate freezing methods on UCB stem cell viability. STUDY DESIGN AND METHODS: Ten UCB units were processed according to standard procedures. After DMSO was added to a final concentration of 10%, each UCB unit was divided into two 25 ml volumes and the bags were placed in metal freezing cassettes. The UCB units were covered by Styrofoam sleeves; for 5 units, one of the 25 ml units was placed directly into a -80oC mechanical freezer and for the other 5 units, one of the 25 ml units was placed into a -140oC mechanical freezer. The corresponding portions of the 10 units were frozen using a Forma Scientific model 1010 controlled rate freezer. Approximately 30 days after freezing, matched units were thawed in a 37oC water bath and tested without washing. Total Nucleated Cell (TNC) count, total CD34+ cell count, 7-AAD viability, and colony forming unit (CFU) assays were performed on fresh and cryopreserved/thawed samples. Statistical pair analysis using student-t test was performed to determine any statistically significant differences among freezing methods. Statistical significance for the student-t test was set at p less than 0.05. RESULTS: Analyses indicated that all three methods produced similar cell viability as measured by TNC recovery, total CD34+ cell counts, 7-AAD viability, and CFU assays. Evaluation of fresh and frozen samples did not indicate that controlled rate freezing produced a better quality product after thawing compared to two uncontrolled freezing methods. CONCLUSION: All three methods evaluated, controlled rate freezing and non-controlled rate freezing at -80oC and -140oC produced similar results. A large-scale study is indicated to confirm these preliminary results.

Poster number: 31
Ni, Lisa, and Timothy J. Sendera; Applied Biosystems, Austin TX 78744, USA
GeneAssist Pathway Atlas View/Hide Abstract

Poster number: 32
Pimton, Pimchanok, Nidhi Sheth, Anant Chopra, Rosemary Bastian, Anusha Rajan, Anat Kasir, Peter Lelkes. School of Biomedical Engineering, Science and Health Systems Drexel University
Low oxygen tension inhibits endodermal differentiation and enhances mesodermal differentiation. View/Hide Abstract

Poster number: 33
Puri, Nitin, Xiaohui Wang, Rajeev Varma, Chris Burnett, Lesslie Beauchamp, Diana M Batten, Vivian Sule, Michelle Young, Kathy Latham, Tim Sendera, David Dorris, Susan Magdaleno; Applied Biosystems, Austin TX 78744, USA
Next-Generation siRNAs: Combining Support Vector Machine Learning with Novel Chemical Modifications for More Consistent siRNA Performance in High-Content Screens View/Hide Abstract

Poster number: 34
Qi, Yanmei, Xiaowen He, Jie Liu, Alan M. Graham and Shaohua Li Division of Vascular Surgery, Department of Surgery, UMDNJ-Robert Wood Johnson Medical School, New Brunswick, NJ 08903
ILK interactions with PINCH and paxillin regulate endoderm differentiation View/Hide Abstract
Integrin-linked kinase (ILK) is an integrin-binding protein that possesses serine/threonine kinase activity. In traditional cultures, it is recruited to focal adhesions and mediates integrin-dependent cell adhesion and spreading. Our previous study has demonstrated that targeted deletion of the ILK gene in mice leads to early embryonic lethality due to defective germ layer formation. ILK has been shown to interact with multiple signaling proteins including PINCH, paxillin and parvins. However, it remains unknown which interactions are important for normal tissue morphogenesis and function. In this study, we stably expressed flag-tagged wild-type ILK or its mutants lacking the binding site for PINCH, paxillin or beta-parvin in ILK-null embryonic stem (ES) cells. ES cell-differentiated embryoid bodies were employed to examine the role of the ILK interactions in epithelial morphogenesis. We found that ILK ablation in EBs impaired endoderm differentiation as manifested by formation of multi-layered endoderm cells and clustering of the cells at one pole on the EB surface. Transfection of the mutant EBs with wild-type ILK completely rescued the endoderm phenotype. By contrast, expression of the PINCH- or paxillin-binding mutant ILK had no effect. Interestingly, ILK that lacks the kinase activity or beta-parvin binding site also restored endoderm differentiation to nearly normal levels. These results indicate that the ILK binding to PINCH and paxillin is essential for normal endoderm differentiation. To explore the underlying mechanisms, we immunostained EBs with the 9EG7 monoclonal antibody that recognizes the active form of integrin beta1 and the HMb1-1 monoclonal antibody that reacts with both the active and inactive forms. We showed that recruitment of active integrin beta1 to the endoderm-basement membrane adhesion site was significantly reduced in the absence of ILK. This was accompanied by diminished assembly of adhesion complexes at the basement membrane zone. These defects were completely corrected by transfection with wild-type ILK but not the mutant ILK that are unable to bind to PINCH and paxillin. Our data suggest that the ILK interactions with PINCH and paxillin may regulate integrin activation to promote endoderm differentiation.

Poster number: 35
Ricupero, C., H. Li, M. Grumet, R. Hart, Dept of Cell Bio & Neuroscience, W. M. Keck Ctr. for Collaborative Neuroscience, Rutgers Stem Cell Research Center, Rutgers University., Piscataway, NJ
Identifying epigenetic regulatory mechanisms in neural restricted precursors derived from the cortex and ganglionic eminence View/Hide Abstract
Epigenetic regulation is essential for differentiating neuronal and glial precursors. Epigenetic modifications such as histone methylation have been linked to cell fate; however the underlying mechanisms controlling the placement of epigenetic marks are still largely unknown. We predict that specific epigenetic marks are reliable indicators for potential gene regulation, thereby revealing the programming of ultimate cell phenotype. To test this hypothesis, 19 neural precursor clones were isolated and immortalized from the E14.5 rat cortex and ganglionic eminence. Isolation at this key developmental stage provided multipotential, neuronal and glial restricted precursors. Gene expression analysis of a differentiated time course was performed using 5 clones representing each restricted phenotype. Results show clear separation of phenotype at key neuronal and glial genes such as Mash1, Vgat and Cystathionine Beta Synthase. However, when focusing on mRNA levels in an undifferentiated state, separation of phenotype is not as clear. To test our prediction that chromatin state in undifferentiated precursors may reveal downstream gene expression, we focused on trimethyl histone modifications linked to gene activation or repression. Clones exhibit distinct marks in specific promoters related to neural differentiation. For example, in neuronal clone Ge6, promoter sequences upstream of the neuron-specific transcription factor MASH1 (Ascl1) and the Neuregulin receptor Erbb4 displayed high levels for a marker associated with gene expression (H3K4me3) and lower levels for a marker associated with gene repression (H3K27me3). In contrast, glial-restricted clone Ge2 showed an inverse relationship displaying increased levels of H3K27me3 and low H3K4me3. This suggests that cell fate may already be predetermined through chromatin modifications in undifferentiated cells. Results from assaying other transcription factor promoters, including members of the DLX and Sox families, provide additional clues for decoding differentiation potential. Our goal is to discover predictive histone modifications at key regulatory genes responsible for downstream cell fate decisions.
NSF IGERT on Integrated Science and Engineering of Stem Cells

Poster number: 36
Schein, L.A., RWJMS DNA Core Facility, UMDNJ, Piscataway, NJ
RWJMS DNA CORE FACILITY PROVIDES FAST AND ACCURATE DNA SEQUENCING AND GENETIC ANALYSIS SERVICES. View/Hide Abstract

Poster number: 37
Seita, Y., Treff, N., and Lu, C-W
Embryonic stem cell modeling early for early trophoblast function View/Hide Abstract
Trophectoderm is the first tissue differentiated from a mammalian embryo. Simultaneously formed with the inner cell mass, cells within the trophectodem give rise to trophoblalsts, the cells essetial to the structure and function of placenta. As the tissue supporting development of the fetus to term, pathogenic condition of placenta is the major cause of perterm complications, including platenta abrupta, preeclampsia, placental insufficiency. The nature of human trophectoderm formation and maintenance has not yet been investigated, particularly in the early developmental stage. Our goal is to test the hypothesis whether trophoblasts formed from differentiating human embryonic stem cells mirrors the process of trophectoderm development in vivo. Human embryonic stem cells H9 and HROSA26-tdRFP are both competent for BMP4-induced trophoblast differentiation. In the presence of 20ng/ml of BMP4, the hES cell morphology transit rapidly into a flattern epithelium like cell type, with clear cellular boundaries indicative of cytokeratin expression. Gene expression in these cells was analyzed by semi-quantitative PCR, showed an effective induction of early trophectoerm transcription factors, including CDX2, AP2g, EOMES, GCM1 and HAND1. Differentiation toward other embryonic lineages (ectoderm, endoderm and mesoderm) all seem compromised in the presence of BMP4. These trophoblasts also express a panel of pregnancy associate hormones, including TRH, GnRH, CRH, hCGa and hCGb, indicating the full differentiation into functional trophoblasts. Pluripotent stem cell markers, including OCT4, SOX2 and NANOG, persistantly express in the BMP4 induced differenitation even through the 9th day post BMP4 induction. It is consistent to the bull-eye colony morphology with a core of cells reflective to the differentiation signal. By plating accutase-treated single cells, the effect of trophoblast induction became homogenous and efficient. Our observation suggests that early trophectoderm differentiation of human embryonic stem cells involving expression of a panel of transcription factors known essential for mosue trophectoderm formation. Additionally, we have also seen that trophoblasts formed from human embryonic stem cells recapitulates the physiological functions of
New Jersey Commission on Science and Technology Living Free for Research Foundation

Poster number: 38
Senthilnathan, Vijayalakshmi, Devi Arikketh, Mohandas PVA , Omana A.Trentz. MIR (MIOT Institute of Research), MIOT Hospitals Chennai, India.
Expression of osteocalcin and nucleostemin in Osteoblasts Derived from Adipose Tissue (ADOB) and Osteoblasts (OB) from cancellous bone. View/Hide Abstract
Introduction:Utilizing the multipotentiality of adult mesenchymal stromal cells, we have succeeded in culturing osteoblasts from adipose tissue (ADOB) and the study provides a comparitive analysis of primary osteoblasts from the iliac crest (OB) and osteogenic differentiated cells from adipose tissue by analyzing the pattern of expression of osteoblasts specific proteins. Methods:We harvested cells from 47 patients, undergoing selective surgery with the patient’s consent. Osteoblasts from cancellous bone were harvested with α MEM medium and ADOB with osteogenic medium, (α MEM medium supplemented with 0.1 μM-dexamethasone and 10mM β-glycerophosphate) and 10% FBS, ascorbic phosphate, and antibiotics/antimycotic. Results:The osteoblasts cultured from adipose tissue (ADOBs) were 100% confluent on day 7, while primary osteoblasts reached 100% confluence on day 10. Characterization of osteoblasts from cancellous bone and ADOB were confirmed by the expression of phenotypic markers of proteins like osteocalcin, ALP, Collagen Type I and Cbfa1 using RT-PCR, Western blot and immunocytochemistry. Although these proteins are expressed in the ADOB cells, the expression of osteocalcin was not similar to the expression in the osteoblasts from iliac crest which expressed prominently the osteogenic proteins specific to osteoblasts. Osteocalcin expression was studied by immunohistochemistry, RT-PCR and these results in both the cells showed variations which further questions the role of adipose tissue as a potential source for multipotent cells for osteogenic differentiation and whether it can successfully used in regenerative medicine. Nucleostemin, the marker gene of Adipose Derived Stem Cells (ADSC) also showed variation in its expression pattern. Nucleostemin has been characterized as a nucleolar protein involved in cell proliferation regulation in adult stem cells, embryonic stem cells and cancer cells. In our study in both the osteoblasts and ADOB cells, cytoplasmic expression along with nuclear expression of nucleostemin was observed in immunohistochemical analysis. This result is in striking contrast to that observed in other studies and thus begins a new debate for the possible reason for its expression in cells other than adult stem cells and cancer cells.
Thanks to MIOT hospitals, India.

Poster number: 39
Settineri, Tina, Shiaw-Min Chen, Chad Mooney, Jane Kuo, Julie Nguyen, Elana Swartzman, Mark Shannon, David Ruff
An integrated quantitative PCR approach for monitoring gene and protein expression in human pluripotent and differentiated NTERA2 cells View/Hide Abstract
The correlation of gene and protein expression changes in biological systems has been hampered by the need for separate sample handling and analysis platforms for nucleic acids and proteins. In contrast to the simple, rapid and flexible workflow of quantitative PCR (qPCR) methods, which enable characterization of several classes of nucleic acid biomarkers (i.e. DNA, mRNA and microRNAs), protein analysis methods such as Western blotting are cumbersome, laborious and much less quantitative. However, TaqMan protein expression assays, which use the proximity ligation assay (PLA) technology, now expand the range of qPCR applications to include the detection of proteins through the amplification of a surrogate DNA template. Here we describe an integrated qPCR approach for measuring relative changes in gene and protein expression from the same starting sample and on a single analytical platform that pairs TaqMan gene expression assays with TaqMan protein expression assays. To validate this approach, we used NTERA2 cells as a model system for studying changes in gene and protein biomarker (e.g. OCT3/4, NANOG, and LIN28) expression in response to trans-retinoic acid induction. Changes in protein and mRNA expression were generally similar in direction and timing. For example, decreased levels of OCT3/4 and NANOG protein and mRNA were evident at day 3 post-induction and showed a continued decrease at day 5. Our results also confirmed a correlation between reduced levels of LIN28 protein and increased expression of mature Let-7 microRNAs. Taken together, these results demonstrate the enormous potential of an integrated qPCR approach for analyzing relative changes in gene and protein expression, and furthermore, reveal dynamic changes in the expression of key proteins and microRNAs implicated in the transition from stem cell renewal to differentiation. These data establish a general paradigm for studying the relationship between the stem cell transcriptome and proteome.

Poster number: 40
Smith, Shannon M. Department of Cell and Developmental Biology Rutgers University, Piscataway NJ; Doh, Sung Tae and Cai, Li Department of Biomedical Engineering, Rutgers University, Piscataway, NJ
Regulation of CD44 expression in breast cancer stem cells View/Hide Abstract
In the last decade, it has been found that a small percentage of cells present in heterogeneous tumor populations possess properties similar to those found in stem cells. These cancer stem cells (CSCs) retain the ability to form new tumors through rapid division and differentiation. Studies have found that CSCs are immune to many cancer therapies and thus our current treatments must change. CD44, a cell surface glycoprotein, is involved in many cellular processes including cell adhesion, migration, and cancer metestasis. CD44 expression is upregulated in breast cancer stem cells (BCSCs), suggesting it may play a role in tumorigenesis and metestasis. However, the mechanism for the regulation of CD44 expression is not well understood. Here we have identified a number of evolutionarily conserved regions within and surrounding the CD44 gene that may play a role in regulating its expression in BCSCs. Conserved regions were isolated from a mouse genome and tested for their ability to direct gene expression using a reporter assay system containing a minimal β-globin promoter and a GFP reporter. These reporter assays were performed in both mouse and human cell lines due to their variable levels of CD44 expression. Results show that three of the 14 conserved regions tested have the ability to drive GFP expression in vitro in both mouse and human cells and GFP expression appears to be dependent on endogenous levels of CD44 expression. Analysis of these regions indicates a number of potential transcription factor binding sites involved in both cancer and embryogenesis. Using an electrophoretic mobility shift assay (EMSA), we were able to identify key regions of binding activity. Mutant competition assays coupled with EMSAs allowed us to narrow down the region of binding activity and identify NFκB as a potential factor binding to one conserved region. In conclusion, we have identified three novel cis-elements that may play a role in regulating CD44 expression in CSCs. Our results suggest that NFκB may be an import factor in this regulatory function via its interaction with a novel cis-element. The understanding of CD44 regulation should be helpful for the future development of effective treatments targeted at the origin of cancer, cancer stem cells.
Funding for this research is provided by NIH (CA133675) to L. Cai.

Poster number: 41
Swartzman, Elana, Mark Shannon, Shiaw-Min Chen, Chad Mooney, Tina Settineri and David Ruff; Applied Biosystems, Foster City, CA 94404
New Protein Expression Assays using qPCR for the Detection and Relative Quantification of Protein Markers in Stem Cells View/Hide Abstract

Poster number: 42
Tang, Fuchou, Catalin Barbacioru, Yangzhou Wang, Ellen Nordman, Clarence Lee, Nanlan Xu, Xiaohui Wang, John Bodeau, Brian B Tuch, Asim Siddiqui, Kaiqin Lao and M Azim Surani
mRNA-Seq whole-transcriptome analysis of a single cell View/Hide Abstract

Poster number: 43
Thompson, Kevin, Alana Toro-Ramos, Jennifer C. Moore, and Rick I. Cohen Rutgers University, Stem Cell Core Facility, 604 Allison Road, Rm D-251, Piscataway, NJ 08873.
A Novel Gene Expression Platform for Generating Inducible Pluripotent Stem Cells View/Hide Abstract
Genetic reprogramming of human somatic cells into pluripotent or hESC-like cells has attracted much attention as a method to carryout stem cell research without ethical or moral issues and providing an avenue to create immunologically matched patient specific material for future clinical use. The most popular method to reprogram mouse or human somatic cells relies on lentivirus mediated cellular transduction of a set of genes, which may include, Oct4 and Sox2 together with Klf4/c-Myc, or with Nanog/Lin28. This robust system permanently transfers the set of pluripotency genes to a given cell, raising the possibility of transformation or interference with downstream differentiation protocols. More recently, others have demonstrated that non-integrating methods, such as adenovirus, episomal vectors and even transposon-based insertion/deletion, can also be used to generate IPS cells. Further refinement and boost in efficiency was made by inserting Oct4/Sox2/Klf4/Myc in one open reading frame separated by self-splicing motifs named "2A" derived from the Foot-and-Mouth Disease Virus. In our Stem Cell Core Facility, we prepared a novel and inexpensive platform (~$0.50/ml of 108 TFU/ml) to express the above-mentioned single factors, or 4 factor single cistronic self-splicing genes in a non-integrating, and biosafe fashion using hybrid baculovirus vector. Using the pFastbac1 backbone, we eliminated the insect polyhedrin promoter, replacing it with a multicloning site with highly rare cutters. We inserted a robust hybrid CMV-IE1/EF1-alpha promoter, followed by fluorescent reporters (turboGFP, mRFP) fused in frame with a choice of several antibiotic selectable markers (blasticidin-S-deaminase = blast), Neomycin Phosphotransferase II = Neo, Puromycin Acetyl-transferase = puro), followed by a woodchuck post regulatory element (WPRE) and bovine growth hormone poly termination (BGH) sequences. Transfection into HEK293 allowed us to verify functional expression of the new plasmid, after which, we ligated in the sets of single or multi-cistronic genes in tandem with novel fusion proteins of turboGFP or mRFP and blast. The pFastbac1 vectors were tested by transfection followed by immunodetection (IC/Western), and found to efficiently express both the single factors, and the spliceable 4 factor versions of pluripotency genes. We packaged the plasmids into baculovirus, and determined that within 12 hours of transduction, human fibroblast took up the virus, and expressed the genes for up to 2 weeks in culture. In summary, we engineered a biosafe (BL1) non-integrating robust platform for gene expression in human cells using the baculovirus backbone. In the future, we will use this system to create a more clinically relevant system for inducing pluripotency in human somatic cells.

Poster number: 44
Toro-Ramos, A.J., J.C. Moore, M. Swerdel, R.P. Hart, R.I. Cohen
Real Time-PCR Detection of Mycoplasma Contaminated Cell Cultures View/Hide Abstract

Poster number: 45
Tzatzalos, E., S. Doh, J. Forrino, M. Grumet, L. Cai, Department of Biomedical Engineering, Rutgers University, Piscataway, NJ Keck Center for Collaborative Neuroscience, Rutgers University, Piscataway, NJ
Regulation of Notch1 during Neural Development View/Hide Abstract

Poster number: 46
Vitale J (1), Stillwell E (1), Zhao Q (1), Beck A (1), Schneider J (1), Khadim F (1), Elson G (1), Altaf A (1), Yehia G (1), Dong H (2), Liu J (2), Mark W (2), Bhaumik M (3), Grange R (4), and Fraidenraich D (1)* (1) Department of Cell Biology and Molecular Medicine, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, NJ, USA (2) Developmental Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY, USA (3) Department of Pediatrics, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway, NJ, USA (4) Department of Human Nutrition, Foods and Exercise, Virginia Polytechnic Institute and State University, Blacksburg, VA, USA *fraidedi@umdnj.edu
Blastocyst Injection of Wild Type Embryonic Stem Cells Induces Global Corrections in Mdx Mice View/Hide Abstract

Poster number: 47
Vlassov1, A.V., A. Cheng1, M. Li1, Y. Liang2, Y. Wang2, L. Wong2, C. Chen2, and S. Magdaleno1 1Life Technologies, Austin, TX; 2Life Technologies, Foster City, CA
Stem-loop RT-PCR quantification of small RNAs in vitro and in vivo View/Hide Abstract

Poster number: 48
Wagner, Katherine, Thomas Schulz†, Eric Sherrer†, Amanda McLean*, Mary Lynn Tilkins, Sackamone Phommachanh, Allan Robins†, Mohan C. Vemuri Invitrogen Corporation • 1600 Faraday Avenue • Carlsbad, California 92008 • USA † Novocell, Incorporated • 111 Riverbend Rd • Athens, Georgia 30602 • USA * Department of Animal and Dairy Science • University of Georgia • Athens, Georgia 30602 • USA
STEMPRO® hESC SFM: A Novel Serum-Free and Feeder-Free Medium for Culturing Human Embryonic Stem Cells View/Hide Abstract

Poster number: 49
Zhang, Xia, Dai-Tse Wu and Monica J. Roth UMDNJ-Robert Wood Johnson Medical School 675 Hoes Lane, Piscataway, NJ 08854
Targeting transduction of lentiviral particles in embryonic stem cells mediated by stem cell specific markers. View/Hide Abstract
A system to specifically target human embryonic stem cells (hESC) for gene transduction has been developed with high efficiency. In this system, lentiviral particles pseudotyped with modified Sindbis virus Envelopes (M168) containing the immunoglobulin Fc-binding domain were able to target the H9 cells via anti-SSEA4 and anti-CD24 antibodies at a transduction level comparable to lentiviral particles pseudotyped with VSV-G. Transduction efficiency was determined by GFP gene transfer into infected cells using flow cytometry. Prior to infection, H9 cells were treated with Accutase and ROCK inhibitor in order to obtain high amount of viable dissociated cells. At a MOI of 1 ~ 2.5, M168-pseudotyped lentivirus could deliver the GFP gene driven by EF-1α promoter at a level up to 90% via anti-SSEA4 and 70% via anti-CD24, whereas GFP delivery in VSV-G-pseudotyped lentivirus was at a level of 95%. A parallel infection study was performed on mature cell lines including fibroblasts and osteosarcomas and showed dramatically lower infection rates, indicating the distribution of these surface receptors provides the specificity towards hESCs cells. A differentiation assay performed on the infected H9 cells displayed that these cells were capable of differentiating into trophoblasts (Cytokeratin 7+, SSEA-4-, CD24- as well as GFP+). This system may be applied to redirect the gene delivery to defined stages along the differentiation pathway of stem cells. Gene expression in hESC cells is promoter-dependent. GFP under the control of CMV promoter could not be detected in undifferentiated H9 cells but was detected in some of their spontaneously differentiated cells. However, GFP gene could be amplified from the DNA extracted from the undifferentiated H9 cells, indicating that the CMV promoter was silenced in the stem cells resulting the loss of GFP expression. In a comparison experiment of three promoters, CMV, EF-1α and PGK, EF-1α was superior to the others in driving GFP expression in terms of percentage and fluorescence intensity. Interestingly, a long-term culture (longer than 30 days) of infected H9 cells driven by the EF-1α promoter showed no attenuation of GFP signal.

Poster number: 50
Zhao X, G Ren, L Liang, B Zheng, Y Shi, JA Tischfiled, C Shao. Department of Genetics, Rutgers, The State University of New Jersey, Piscataway, NJ
IFNgamma Induces Expansion of Hematopoietic Progenitor Cells View/Hide Abstract

Poster number: 51
Jing Xu1, María Pía Rossi2, Craig K. Griffith3, Jean Schwarzbauer4, Joachim Kohn5, Prabhas V. Moghe1, 2, 3. Department of 1Cell & Developmental Biology, 2Chemical & Biochemical Engineering, 3Biomedical Engineering, 5Chemistry & Chemical Biology, Rutgers University, Piscataway, NJ 08854 4Department of Molecular Biology, Princeton University, Princeton, NJ 08544
Combining Biofunctionalized Nanoparticles and Electrospun Tyrosine-derived Polycarbonate Fibers: a New Mesenchymal Stem Cell Based Approach to Treat Chronic Wounds View/Hide Abstract

Poster number: 52
Mindy Ezra, New Jersey Center for Biomaterials, Piscataway, NJ, USA Rutgers University, Piscataway, NJ, USA Melitta Schachner, W. M. Keck Center for Collaborative Neuroscience, Piscataway, NJ, USA Joachim Kohn, New Jersey Center for Biomaterials, Piscataway, NJ, USA Rutgers University, Piscataway, NJ, USA
Tyrosine-derived Polycarbonate Conduit to Improve Functionality of Peripheral Nerve Regeneration View/Hide Abstract
Once severed, peripheral nerves can regenerate, however often result in poor functional recovery due to their tendency to extend randomly. There is an immediate need to help guide regenerating nerves in order to increase functional recovery. Recently, tubulization, implanting a tube around the nerve gap and bringing the stumps into the tube, has become a popular method to repair severed nerves because it provides direction to axons. Additionally, in mice, the human natural killer cell glycan HNK1 is known to help guide motor axons during growth and regeneration, providing more directional guidance to the nerve. Our approach to enhance nerve regeneration is to fabricate a biodegradable, polymeric scaffold using tyrosine-derived polycarbonates that consists of an outer tube and an inner bioactive component. We fill our tubes with HNK1 grafted to an ECM protein. In the future, we will also deliver a supply of HNK1 to sites of peripheral nerve injury via genetically engineered mesenchymal stem cells. Preliminary studies in which a series of conduits were assessed for their ability to accelerate regeneration in 5mm gaps in the mouse femoral nerve are complete. Following transection, tubes were implanted with either saline, ECM protein, or HNK1 grafted to ECM protein as filler. MSCs secreting HNK1 were not used in this study. Functional analysis was performed weekly in order to evaluate muscle function during gait. Retrograde labeling was performed in order to determine the number of axons that correctly reconnected. Functional results show polycarbonate conduits perform better than control polyethylene conduits. When filled with HNK1 + ECM component, both polymers result in positive recovery. Retrograde labeling for groups with positive functional recovery, shows motor axons properly reconnected to the appropriate motor branch and that HNK1 enhanced functional recovery. We are creating a delivery system for genetically engineered MSCs to produce and secrete there own HNK1 into the wound space. This system will be based on lenti- and adenoviral vectors and expression will be controlled so as to secrete HNK1 only at certain times. MSCs engineered to secrete HNK1 may provide additional direction to regenerating nerve which may increase functional recovery.
CeMBR - Center for Military Biomaterials Research Stem Cell IGERT - Integrative Graduate Education and Research Traineeship

Poster number: 53
Sadik, M.,M.,Shan, J., Lin, H., Mechanical and Aerospace Engineering, Rutgers University, Piscataway, NJ Shreiber, D., Biomedical Engineering, Rutgers University, NJ
Electroporation-Mediated Molecular Delivery View/Hide Abstract

Poster number: 54
Presenter: Dr. Alexandra Vamvakidou-Thomas
Real-Time qPCR Data Analysis using RealTime StatMiner View/Hide Abstract

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