Archives

  • 2018-07
  • 2018-10
  • 2018-11
  • br Materials and methods br

    2018-11-06


    Materials and methods
    Results
    Discussion Currently, a number of cell types such as EPCs, bone marrow mononuclear cells, and mesenchymal stromal daunorubicin are being tested in clinical trials for their efficacy in treating cardiovascular diseases (O\'Neill et al., 2012). Although there appears to be some benefit with adult cell therapy, this appears to be modest, and dependent primarily on paracrine mechanisms (Lasala and Minguell, 2011). Thus, the initial hypothesis that these cells would robustly differentiate into cardiac and vascular cells and directly regenerate damaged tissue has largely been unfulfilled. Human ESCs, and now iPSCs, offer potential alternative cell sources for cell therapy with much greater potential for differentiation and tissue regeneration. As well, derivation of iPSCs from patients with disease represents a valuable tool to better define underlying disease etiology and to screen for new drug therapies. However, the need for biopsy samples to derive patient-specific iPSCs is a major limitation, particularly the access to biopsies from relatives of the patients to act as genetic controls. Thus, the ability to derive iPSCs from blood enables the wider application of this technology and study of a broader range of diseases. Collectively, our data shows that EPC-iPSCs are pluripotent. Further, we have reprogrammed late-EPCs under feeder-free and defined conditions, which should facilitate the progress of ultimately using EPC-derived iPSCs in a clinical setting. However, before this can be achieved, reprogramming of late-EPCs using non-integrating methods would be necessary. In the course of our study, we attempted to reprogram late-EPCs using several non-integrating strategies such Sendai Virus (Macarthur et al., 2012) and episomal vectors (Okita et al., 2011). Using these reprogramming methods we were not able to achieve successful reprogramming (data not shown). In fact, Sendai virus mediated reprogramming killed the late-EPCs, while generating control iPSCs. Despite generating adult dermal fibroblast-derived iPSCs using episomal reprogramming vectors, late-EPCs transfected with these vectors did not proliferate well after switching to the hESC medium at different times post-transfection, suggesting expression of the reprogramming factors was not high enough to induce reprogramming. Thus, further optimization including the use of small molecules or the use of alternate reprogramming methodologies such as mRNA is required for successful non-integrative reprogramming of late-EPCs. Nonetheless, our study is a step forward in utilizing peripheral blood-derived late-EPCs as a cell source to derive feeder-free iPSCs in defined conditions. Late-EPCs represent an ideal cell source for reprogramming because of their accessibility from peripheral blood, their high proliferative capacity, and homogeneous population that can be frozen and thawed (Lin et al., 2011) for future genomic and proteomic analysis. Furthermore, unlike lymphoid lineages, L-EPCs do not undergo V(D)J recombination; therefore iPSCs derived from L-EPC would closer reflect other cell types from the donor which can eventually be applied to understanding diseases/disorders.
    Acknowledgments The authors would like to acknowledge the assistance from members of the Stanford and Stewart labs. This work was generously supported by the following organizations and funding agencies: the Canadian Institutes of Health Research (CIHR) to WLS for an operating grant (MOP-89910) and a CIHR Banting and Best CGS Doctoral Research Award to JLM; the Heart & Stroke Foundation of Canada for a Postdoctoral Fellowship to WYC; the Fonds de la recherché en santé du Quebec to JRL; the Ontario Research Fund (WLS); and the Canada Research Chair program which provides support to WLS through a Tier 1 Chair in Integrative Stem Cell Biology.
    Introduction Pluripotent stem cells hold the potential to generate all cell types in culture, but the development of methods which control differentiation to induce generation of one cell type over another has proved challenging. The differentiation of mouse and human pluripotent stem cells into specific neural subpopulations has been achieved using determined extracellular environments, specific signaling factors, small molecules, co-culture systems, or transgenic modifications (Chung et al., 2002; Friling et al., 2009; Kawasaki et al., 2000; Lee et al., 2000; Martinat et al., 2006; Okabe et al., 1996; Tropepe et al., 2001; Wichterle, et al., 2002; Ying, et al., 2003). However, despite the general progress in differentiation strategies, several aspects are still insufficiently understood, especially when aiming at pluripotent cell-derived neurons for regenerative approaches. For example, lack of synchronicity and purity of the target cell population represent two major limitations. Target cells should fit into a specific developmental window since implantation of pre-differentiated and proliferative cells can give rise to slow-growing tumors (Roy et al., 2006), while cells too advanced in their differentiation may be unable of functional integration (MacLaren et al., 2006). Moreover, the presence of undifferentiated pluripotent cells must be avoided to prevent teratoma formation upon in vivo injection (Bjorklund et al., 2002; Thomson et al., 1998).