Browsing by Subject "Stem Cell Institute"

Now showing 1 - 5 of 5
  • Thumbnail Image
    Item
  • Thumbnail Image
    Item
    Gene expression differences between hemangiosarcoma cells in monolayer and non-adherent sphere culture
    (2011-04-13) Sahli, Nathanael
    The cancer stem cell (CSC) theory argues that tumors have a subset of cells that initiate, maintain, and expand cancer in an affected patient. Experimental support for this theory comes from studies that identified sub-populations of cells in a tumor that have the capacity to evade common cancer treatments such as chemotherapy and radiation. Additionally, these same cells exclusively retain the capacity to initiate new disease in xenograft studies. The study of these evasive cells was initially challenging as they differentiate in standard serum-containing culture medium where they grow as a monolayer. In the past decade, methods for culturing stem cells using a serum-free medium has allowed CSCs to be maintained, where they form non-adherent multicellular spheres. Here, we cultured canine hemangiosarcoma (HSA) in both a multicellular sphere and standard monolayer system to compare gene expression using real time qRT-PCR. In our system, the monolayer cultures are a useful surrogate for differentiated tumor cells (the bulk of the tumor), while the serum-free sphere-derived cells are a surrogate for in vivo CSC. Here, we investigate differences in gene expression between these two cultures systems. The genes chosen for study have been shown to be up-regulated in CSCs from various other cancers, or normal stem cells, with minimal expression in differentiated cells. We found gene expression differences between cultures conditions which will allow it to be utilized in the study of hemangiosarcoma as well as possibly other cancers with a CSC.
  • Thumbnail Image
    Item
    Protection Against Acute Kidney Injury by TUDCA
    (2009-04-08) Ononenyi, Chimezie U.
    Presently there is no therapy for acute kidney injury (AKI). Potentially one can protect kidneys against injury by preventing cell death following AKI. There are two types of cell death that occurs following AKI: necrosis and apoptosis. Necrosis is uncontrolled and synchronous cell death that occurs at the time of AKI. In contrast, apoptosis, or programmed cell death is an asynchronous cell death that continues to occur for days following AKI, thereby providing a window of opportunity for intervention. Tauroursodeoxycholic acid (TUDCA), a bile acid synthesized in the liver, has been shown to be effective by inhibiting apoptosis in rat models of stroke and Huntington’s disease. We hypothesized that TUDCA will be similarly effective in protection against AKI. Accordingly, the goal of this study was to investigate the protective effects of TUDCA in a rat model of ischemic AKI. We induced AKI by bilateral renal artery clamping for 45 minutes. Three rats were given 400mg/kg/day of TUDCA intraperitoneally from day -1 to day 6, while the control animals received the vehicle. We determined kidney functions by measuring blood urea nitrogen (BUN), proliferation by immunohistochemistry for Ki67, and apoptosis by TUNEL assay. Compared to a control, animals that received TUDCA had less severe kidney injury and apoptosis. In conclusion, TUDCA provides protection against acute kidney injury likely by preventing apoptosis.
  • Thumbnail Image
    Item
    ShRNA Knockdown of ID genes in Human Embryonic Stem Cells as a Possible Path Towards B and T Cell Development
    (2009-04-08) Taylor, Russ
    Human Embryonic Stem Cells (hESCs) are pluripotent, self-renewing cells capable of becoming any cell in the human body. Previously, our lab has derived multiple cell types from hESCs, with a particular interest in hematopoietic (blood cell) development. We have been able to successfully derive natural killer (NK) cells, a type of lymphocyte with potent anti-tumor activity. However, to date we have been unable to derive other lymphocytes (T and B cells) from hESCs. Using Umbilical Cord Blood (UCB) as a comparison for early hematopoietic development, we deduced that one possible factor for this difference could be a relatively high expression of the Inhibitor of Differentiation (IDs) transcription factors in hESCs compared to UCB. ID proteins bind to and negatively regulate basic Helix-Loop-Helix (bHLH) proteins responsible for many differentiation programs within the cell. In particular, ID2 and ID3 are known to promote NK cell development and inhibit B and T cell development. My project has been to introduce shRNA constructs into hESCs to inhibit expression of ID2 and ID3 as a means to better promote T and B cell development from hESCs. Two shRNA systems have been designed. The first uses a lentiviral vector in which the shRNA construct is constitutively expressed. The second uses a lentiviral vector containing a CRE-conditional shRNA expression system. We have introduced both of these vectors into Ntera2 embryonal carcinoma cells and hESCs. For the constitutive shRNAs, we demonstrate partial knockdown of ID2 and ID3 via qRTPCR. These constitutive knockdown ID hESCs become more difficult to culture in an undifferentiated state, and over time may have lost some of their potency. This potential problem with constitutive knockdown cells highlights the need for the inducible system, which is in progress. We anticipate that the ability to inhibit ID2/3 expression at specific timepoints of hESCs differentiation into hematopoietic cells could solve this problem and facilitate development of B and T cells from hESCs.
  • Thumbnail Image
    Item
    Spin Embryoid Bodies as an Improved Method of Blood Cell Differentiation in Human Embryonic Stem Cells and Induced Pluripotent Stem Cells
    (2010-04-21) Armstrong, Rebecca
    Two types of cells have been characterized as pluripotent stem cells: human embryonic stem cells (hESCs) and the more recently described induced pluripotent stem cells (iPSCs). Pluripotency means that these cells have the ability to form all the cells and tissues of the human body. hESC and iPSC differentiation into blood cells is particularly useful in studying the mechanisms of blood diseases and cell transfusion therapies. To better understand the potential of these cells to specifically produce blood cells, it is necessary to define an efficient and reproducible system of differentiation. Previously, our and other groups have used a stromal cell co-culture method to support blood development from hESCs. In these current studies, we are employing a stromal-free and serum-free differentiation method that may facilitate clinical translation of hESC- and iPSC-derived cells. This method involves forced aggregation of defined numbers of undifferentiated hESCs or iPSCs in 96-well plates by centrifugation to form embryoid bodies (spin EBs) of a uniform size. We examine this method’s potential to derive blood precursor cells and mature blood lineage cells from both hESCs and iPSCs.