Publication Type: Poster
Review Method: Peer Reviewed
Abstract: Progress towards embryonic stem cell (ESC) therapies is hampered by the occurrence of teratocarcinomas, tumors derived from undifferentiated ESCs, and by a lack of understanding of the host conditions that promote integration of transplanted cells. Isolation of ESC differentiated derivatives, free of contaminating undifferentiated ESCs, prior to transplant could decrease the risk of tumor formation. Our previous studies involving transplantation of ESC derived neural stem cells (NSCs) into the kainic acid (KA) induced seizure mouse model of temporal lobe epilepsy (in SCID mice) showed that transplanted cells migrate to the upper blade of the dentate gyrus (DG), where they incorporate into the granule cell layer and express local molecular markers. However, prior seizure experience was required to eliminate teratocarcinoma formation as tumors were frequently seen in un-seized control animals (Carpentino et al., 2008). Working with the Sox1-GFP mouse ESC line, which has a GFP reporter under the control of the promoter of the neural specific gene Sox1, we can derive NSCs that can be isolated by FACS to reduce the presence of undifferentiated ES cells. These cells were injected into the CA3 region of hippocampus and their fate was observed 4 weeks following transplant. Thus far, we have only observed one case of tumor formation out of 12 animals with no prior seizure experience using these cells. These preliminary studies suggest that seizures are not required for cells to incorporate into the DG and that FACS enrichment of NSCs reduces teratocarcinoma formation. We are currently working on similar protocols to prepare derivatives of the H1 and H9 human ESC lines for transplantation studies.

Publication Type: Poster
Abstract: This study examines the high school and post-high school educational pathways of over 91,000 1996-97 Florida public high school graduates. We construct high school course-taking and achievement Science, Technology, Engineering, and Mathematics (STEM) pathways using mathematics and science courses and use these pathways to predict future college and university enrollment and non-STEM and STEM degree attainment.

High School course-taking and achievement are key factors in enrollment and employment trajectories in the first two years after high school and enrollment through 2004. Students who achieved a C or above in Calculus and Physics while in high school are on a STEM pathway and are more likely than lower achieving students to enroll in a four-year university, obtain a bachelor’s degree, and are more likely to obtain a STEM degree in Physics, Chemistry, Biology, Agricultural Sciences, Engineering, Mathematics, or Computer Sciences major.

Students who achieved a C or above in courses at or above the Algebra II and Chemistry levels are on a college pathway out of high school have higher degree attainment than borderline pathway students who only completed Algebra II or Chemistry, but both college and borderline students had favorable enrollment outcomes.

Students who did not successfully complete Algebra II or Chemistry generally went into the workforce or attended community college after high school. Few students enrolled in a university or out of state institution and very few obtained a bachelor’s degree.

Publication Type: Conference Paper/Unpublished Manuscript
Review Method: Peer Reviewed
Abstract: Religious fundamentalists have been vocal in their opposition to embryonic stem (ES) cell research. In this paper, I investigate the influence one’s religious beliefs have on political behavior. Specifically, the paper explores the impact Christian fundamentalism has on opposition/support for the 2006 Missouri Stem Cell Research and Cures Initiative. Using the Roper Center for Public Opinion’s exit polling, the findings suggest that white, conservative, Republicans who self-identify as Evangelical Christian and attend religious services at least once a week were significantly more likely to vote against the ballot measure in the 2006 mid-term election. Therefore, political scientists should consider the influence religion has on voting behavior, particularly concerning controversial biomedical research using human embryos.

Publication Type: Poster
Review Method: Peer Reviewed
Abstract: Abstract
Over the past four decades considerable research has been conducted in animal stem cell identification. In 1998, the attention shifted to the identification of human embryonic stem cells and adult stem cells. Research scientists from UK, USA and Korea used different methods like fluorescence-activated cell sorting (FACS) and flow cytometry with a blue laser to isolate the different markers associated with different embryonic and human cells. Over the years the need to properly identify these stem cells while they differentiate into specific cell characteristics is increasing, but there is an urgent need to overcome the hurdles and pitfalls of improper identification that may result in tumorous results. We propose a method that employs Wavelet Transform Signal Processing methods to increase the accuracy in identification of stem cells using near infrared (NIR) cameras. Markers corresponding to different proteins that generate unique spectral characteristics are more accurately identified with this novel system. With this new system medical researchers can now use precise information to seed a fully cultivated set of stem cells in appropriate human organs to cure dreaded diseases like Parkinson’s, diabetes, coronary arterial damage of the heart and cancer. The entire system is combined into a hardware and software system that will ultimately create a system called as Identification of Human Embryonic Stem Cell using Signal Characteristic Analysis with the acronym of hE[SC]2AN. It is imperative to note that this project does not delve with the growing of cell cultures or reinventing the biological process of differentiation of stem cells. The proposal system serves as a tool for accurate identification of stem cell markers using state-of-the-art imaging techniques. Recent exercises to evaluate our techniques have revealed the success of our imaging techniques for differentiating stem cells as discussed in the sequel. Also recently researchers from UCLA, UC Davis have developed a superior near infrared-emitting quantum dots technique for in vivo imaging and have been working with research collaborators at Stanford University to perform in vivo imaging with these probes in mice. Their technique uses NIR methods which is also the basis for this methodology.

Advanced Bio-Instrumentation to Identify and Isolate Stem-Cell Lineages
Stem cells have unique developmental properties that make them critical for new cell-based therapies involving transplantation needed to treat degenerative and genetic diseases like Parkinson, cardiac disorders and cancers. Stem cells can be isolated from different embryonic and adult tissue sources and different cell and molecular factors have been used to produce different cell types. Current efforts are focused on characterization of cells at different times during differentiation, identifying different regulatory factors responsible for expression of different cell populations and developing methods and instrumentation to identify and subsequently isolate specific cells. The proposed research addresses the need for improved bio-instrumentation that can be used to both analyze and manipulate human stem cell populations for treating patients with a wide range of clinical diseases. Recently, there has been a remarkable advance in research focused on understanding how different cell and molecular factors control proliferation and differentiation of embryonic and adult human stem cells. However, it is critical that we also develop new imaging, detection, identification, sorting and processing technologies specifically for human stem cell populations before clinical therapy can be successful. Among the most common methods for identifying stem cells are indirect procedures which are inefficient, time consuming and not very sensitive. They rely on analyzing cell progeny after the differentiation of the stem cell lineages. The problem is that cells are often phenotypically unstable and expression is transient under different growth conditions. The development and application of powerful new imaging methods has become essential to identifying specific cell types appearing during differentiation. Our approach is to apply near infrared (NIR) imaging employing Wavelet Transform Signal Processing methods to increase signal strength and accuracy. The advantages of this method are the following:

– Greater signal to noise ratio: Use of novel dyes for labels that do not overlap spectrally with visible dyes Very low autofluorescence/background Low energy excitation wavelengths cause less damage than visible dyes The proposed work plan involves designing and constructing an imaging system using the SC 6000 NIR camera system coupled to an image processor to collect digital images of fluorescently labeled human mesenchymal stem cell lineages at different stages during differentiation. Images will be processed and stored in a database which can be evaluated with new signal processing protocols using Wavelet Transform methodology to resolve small differences between phenotypes of cells. Our rationale is that new sensitive imaging bioinstrumentation coupled with knowledge about expression of lineage specific biomarkers can be used for the exact identification, purification and processing of specific cell populations. We hypothesize that this approach can increase the yield of specific stem cells for transplantation and decrease contamination by other cell types, especially oncogenic lineages. We believe it is critical that the development of advanced instrumentation for identifying/isolating cells proceed pari passu with the methods to grow cells and characterize different regulatory factors. We will be testing multiple fluorescent labels to increase quantum yield and improve imaging sensitivity in combination with different biomarkers to enhance cell identification so specific cell populations can be discriminated during growth in culture.

References
[1] “Autocrine growth of small cell lung cancer mediated by coexpression of c-kit and stem cell factor”, GW Krystal, SJ Hines and CP Organ Department of Medicine, Medical College of Virginia, Richmond, USA.
[2] “Novel Stem Cell Technology Leads To Better Spinal Cord Repair”, University of Rochester Medical Center report - The University of Rochester Medical Center and Baylor College of Medicine, Houston, collaborated on the work, April, 2006 issue of the Journal of Biology.
[3] Cells discovered at MIT may play role in lung cancer, June 29, 2005, article published and researched on Internet.
[4] Hemchandra Shertukde, “Near Infra Red Imaging using state-of-the art cameras and Wavelet Transform tracker for Embryonic Stem Cell Identification.” Proceedings, workshop on FLIR applications at the Inframation Exposition, Las Vegas, NV, October, 2006.
[5] H. Shertukde, ‘Signal Processing Tools for Identification of hESC’, Mini Symposium on Human Embryonic Stem Cell Research and Its impact on the State of Connecticut, UT 320, CETA, UHART, March 17, 2006.
[6] Mills, E., LaMonica, K., Hong, T., Pagliaruli, T., Mulrooney, J. and Grabel, L. “Roles for Rho/Rock and vinculin in parietal endoderm migration. Cell Communication and Adhesion. (in press)
[7] Hemchandra Shertukde, Rekha Shertukde, Karan Shertukde “Near Infra Red Imaging using state-of-the art cameras and Wavelet Transform tracker for Embryonic Stem Cell Identification.” Proceedings, Ist CT Symposium on Stem-cell research, Farmington, CT, September 17, 2008

Publication Type: Poster
Review Method: Peer Reviewed
Abstract: The ability to readily derive neural stem cells (NSCs) from human embryonic stem cells (hESCs) is a necessary step in generation of transplant material for the potential treatment of neurodegenerative diseases. Standard NSC derivation protocols utilize an embryoid body (EB) intermediate followed by extended culture, which can take from 30 to 60 days, to direct differentiation into neural progenitors. We have modified established neural differentiation protocols to produce a more direct and efficient method of generating enriched populations of NSCs. Our EB-free protocol utilizes Noggin, a BMP-antagonist and well known neural inducer, for directing differentiation of both H1 and H9 hESC lines toward a neural cell fate, while discouraging the development of non-neural cell types. On Day 0, cells are passaged manually onto a mitomycin-C treated mEF feeder layer into N2B27 neurobasal medium supplemented with Noggin and are maintained in this medium for 10 days. As the mEF feeder layer undergoes cell death, hES colonies expand both from their center and at points around their periphery, as individual cells take on a bipolar morphology. On D10, the cells are passaged manually onto laminin-coated substrates. Following the transition from feeder layer to laminin, the cell population undergoes massive cell death. By D12, a subset of the NSC population begins to assemble into immature rosettes; bipolar cells arranged radially, a structure frequently observed during ESC neurogenesis. At this stage, cells arranged in rosettes, as well as others in the surrounding monolayer, express the neural progenitor transcription factor Pax6, as well as the neural stem cell markers Musashi1 (Msi1) and Nestin. By D14, the rosettes have matured and individual cells take on a columnar morphology and robustly express Msi1 and Pax6. A subset of cells, beginning at D12 and peaking at D14, also express Forse1, a surface epitope expressed by progenitors with anterior CNS identity. Preliminary data also suggest that the midbrain marker Engrailed1 (En1) is expressed by a few cells within the rosette core, as well as a large number of cells surrounding the rosette, but is noticeably absent from the columnar cells of the rosettes. These data suggest the protocol can produce neural progenitors with broad regional specificities, a conclusion we are currently examining in more depth.