Lysosomal Cystine Enhanced Apoptosis In Cultured Human Mesenchymal Stem Cells
Cystinosis Research Foundation Six Month Progress Report: Jess G Thoene, M.D.
Lysosomal Cystine Enhanced Apoptosis in Cultured Human Mesenchymal Stem Cells
7 May 2007
Funds from CRF have enabled re-establishment of a functional research lab at the University of Michigan. Major equipment has been made available by the Division of Pediatric Genetics, allowing the bulk of the CRF funds to be reserved for operational expenses as detailed in the application.
To date, the following has been accomplished:
Occupancy of Medical Science II Rm 3725- This space was completely vacant, hence all lab equipment and supplies had to be ordered and installed. These include: Laminar flow tissue culture hood, CO2 incubator, inverted microscope, centrifuges, pipettes, computers, telephones. Access to an adjacent liquid scintillation counter was obtained.
Authorization to order, posses and use radioisotopes was achieved.
Cultures of normal and cystinotic human diploid fibroblasts were ordered and received from the Coriell Institute and are now in repertoire in this lab with back- up cultures frozen in liquid N2 canisters and sub-zero freezers. These lines are:
GM00008 CYSTINOSIS, NEPHROPATHIC; CTNS
GM00010 APPARENTLY HEALTHY FETAL TISSUE
GM00046 CYSTINOSIS, NEPHROPATHIC; CTNS
GM00090 CYSTINOSIS, NEPHROPATHIC; CTNS
Apoptosis has been induced in these lines with the protocols adopted at Tulane, using 30 ng/ml of TNF alpha and 2.5 µg/ml of actinomycin D. At 16 hours the cells are clearly apoptotic.
100mm plates of cystinotic and normal cultured fibroblasts have been treated to induce apoptosis, harvested, and the cell pellets from two separate experiments taken to the University of Michigan Proteome Consortium Laboratory where they will be treated with ICAT reagents (Isotope-Coded Affinity Tags). These molecules possess an affinity tag, an isotope tag and a protein-reactive group for attachment to cysteine groups in proteins. A non-deuterated and deuterated pair of ICAT reagents are used to determine the relative levels of proteins in complex protein mixtures. By using this mixture pre- and post reduction of the proteins harvested from normal and cystinotic cells that either have, or have not been treated to induce apoptosis, we should be able to determine which proteins were cysteinylated by the cystine released from cystinotic lysosomes. The chemistry is complicated, and initial results will take several weeks to obtain.
A recognized potential obstacle in this process is inadequate instrument sensitivity for the available sample size. Only about 15-20 per cent of cells undergo apoptosis under these circumstances, and the important proteins that are cysteinylated are of low abundance to begin with, hence the labeled proteins may be at or beneath the limit of detectability of this method. If this is the case, there are two options: 1) Increase the number of cells harvested so that total protein abundance is increased 2) Employ FACS to sort the cells using an apoptotic marker, so that 100% of cells studied are undergoing apoptosis.
When conditions are defined which allow us to identify cysteinylated proteins using normal and cystinotic fibroblasts, as herein described, we will obtain hMSC from Tulane to conduct the stem cell portion of the experiments, as described in the application.
