Experimental autoimmune encephalomyelitis (EAE) is an autoimmune inflammatory disease of the CNS that is mediated by T cells and macrophages and represents the paradigmatic model for multiple sclerosis (MS). In EAE, therapeutic approaches targeting T cells have been successfully utilized leading to immunosuppression or tolerance. It has been recently shown that mesenchimal stem cells (MSC) isolated from C57BL/6J mice are an effective treatment of EAE induced by the encephalitogenic peptide pMOG 35-55. In this project we aim to track and evaluate the molecular changes the MSC acquire through several in-vitro passages that determine their therapeutic potential. Elucidation of the molecular pathways involved in this process will allow us to formulate better models of disease progression and curative strategies.
Specific aim:The specific aim of this proposal is to dissect the molecular pathways that mesenchimal stem cells undergo during in-vitro treatment and confer them the therapeutic potential to cure EAE. To accomplish this aim, we will purify and grow murine MSC under the same conditions used for therapy as described in Zappia et al (In press. Brain, 2005) and obtain samples at several stages of the culture. RNA will be purified from each sample and profiled using Affymetrix microarrays. Initial bioinformatics analysis will include data transformation, normalization and filtering. A second stage analysis will involve time-series analysis by ANOVA and dynamic events modeling using neural networks.
Stem cells are now viewed as potential source of cells for almost any tissue due to their postulated capacity to give rise to virtually any type of cell. Among them, mesenchymal stem cells (MSC), can extensively proliferate in vitro and differentiate under appropriate conditions in bone, cartilage, and other mesenchymal tissues. It has been also reported that MSC can differentiate into other cells types including neuroectodermal cells. Furthermore, upon tissue injury MSC have been shown to migrate into the damaged brain. These results suggest that MSC could provide an ideal cell source for repair of injured organs including the central nervous system (CNS). In addition to their plasticity, MSC have been recently demonstrated to suppress several T lymphocyte activities thus exerting an immunoregulatory capacity both in vitro and in vivo. The mechanisms mediating such effect are only partially understood. We hypothesize that an analysis of how MSC acquire their therapeutic potential in vitro by expression profiling in a time series fashion will allow us to dissect the involved mechanisms.
Before arriving to our lab in UCSF, samples will be processed as follows: Bone marrow from 6-8 weeks old C57BL/6J mice will be flushed out of tibias and femurs. After two washings by centrifugation at 1500 rpm for 5 min in PBS (Sigma Chemical Co., St. Louis, MO, USA), cells will be plated in 75 cm2 tissue culture flasks at the concentration of 0.3-0.4 x 106 cells/cm2 utilizing Murine Mesencult as medium (Stem Cell Technologies Inc., Vancouver, BC, Canada). Cells will be kept in a humidified 5% CO2 incubator at 37°C for about 4-5 weeks, when only adherent cells will be collected following 10 min incubation at 37°C with 0.05% trypsin solution containing 0.02% EDTA. After the first cut and for the subsequent 4 or 5 passages, cells will be plated in 25 cm2 flasks at 1.2-2.0 x 104cells/cm2 . For the following passages cells will routinely seeded at 4-10 x 103cells/cm2 reaching confluence only after 4-5 days. Cells will be collected at selected timepoints and immersed in TRizol reagent and shipped to UCSF. For the initial stage of this project cell will be collected at the following 5 time points:
T0: Bone marrow aspirates (uncultured)
T1: pre-mesenchymal stem cells (some cut before the population completely subtracted of Hematopoietic stem cells)
T2: Mesenchymal stem cells (in vitro assessed, functionally and phenotipically)
T3: Mesenchymal stem cells biological active in vivo
T4: Mesenchymal stem cells at the passage in vitro next to the one active in vivo
T5: Mesenchymal stem cells no more active in vitro neither in vivo
Once in our lab, RNA will be extracted using the Trizol reagent (Invitrogen) and further purified by the RNeasy kit (Qiagen). Sample labeling will be performed according to the array manufacturer (Affymetrix) instructions. Subject to initial results, we plan to add intermediate timepoints in subsequent stages.
Keywords: time-course
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