Results br Discussion In this
Discussion In this study, we described the differentiation of hiPSCs into efficient functional ECs and compared them with hESC-ECs. We have found that hiPSCs, similar to hESCs, can be differentiated into ECs by the use of a sequential combination of FLK1-positive (EPC marker) and CD31-positive (late EC marker) sorting. These ECs show typical endothelial cobblestone morphology, express lineage specific markers (CD31, CD144, and CD146), and can take up DiI-ac-LDL as well as form endothelial tubes on Matrigel. These results are in agreement with other recent reports on the differentiation and functionality of ESC and iPSC-derived ECs (Kaufman et al., 2004; Kim and von Recum, 2009; Levenberg et al., 2002; Li et al., 2007, 2011; Rufaihah et al., 2011; Sone et al., 2003; Yamashita et al., 2000). We induced SSc in a mouse model by administering daily subcutaneous injections of bleomycin. The model was characterized by accumulation of collagen bundles, increased mast cell numbers (particularly in the edematous phase), observation of degranulated mast cells, and the release of TGF-β1 as previously noted (Jinnin et al., 2004; Li et al., 2007; Reinecke and Murry, 2002; Yamamoto, 2010). We observed that transplanted hiPSC-ECs showed homing around damaged vessels, with improvements in collagen deposition, the numbers of total and degranulated mast cells, in addition to superficial injury healing four weeks post-transplantation. The beneficial effects of hESC-ECs and hiPSC-ECs in the setting of myocardial or limb ischemia have also been reported (James et al., 2010; Kane et al., 2010; Levenberg et al., 2002, 2010; Li et al., 2009; Nourse et al., 2010; Rufaihah et al., 2010; Sone et al., 2007). The mechanism for this improvement remains to be determined; however, it may be based on the following: (i) attendance of a subpopulation of mature or precursor THZ531 that survive, migrate, and differentiate to ECs, (ii) paracrine effects, and/or (iii) the prevention of mononuclear cell infiltration, thus resulting in improvement of symptoms (Fig. 9). We have observed the incorporation of hiPSC-ECs into existing damaged vasculature. Poor donor cell survival of transplanted hiPSC-ECs has been reported in other studies as indicated by serial histology, the Taqman Sry PCR technique, or TUNEL apoptosis assay (Li et al., 2007; Reinecke and Murry, 2002). This may be related to stimulation of the immune system of animals by human cells or may be related to inappropriate receipt of a signal required for efficient integration of hiPSC-ECs into mouse vasculature. Alternatively, it is likely that the injected cells have a paracrine effect. Recently, it has been demonstrated that transplanted hiPSC-ECs in an ischemic limb model caused secretion of the angiogenic cytokines and growth factors, including angiopoietin-1, vascular endothelial growth factor-A and -C, and platelet-derived growth factor-AA (Rufaihah et al., 2011). However, additional studies are necessary to test our hypothesis. In addition, the perivascular infiltration of mononuclear cells (MNC) is associated with increased collagen synthesis in adjacent fibroblasts (Jinnin et al., 2004). Therefore hiPSC-EC transplantation, homing, and incorporation in the reconstruction of damaged vascular barriers can prevent MSC infiltration, which will lead to the prevention of extra collagen synthesis and subsequent occurrence of fibrosis by intervening in the fibrotic process. One of the limitations of our study is that we focused on one hiPSC line; additional research on more hiPSC lines is necessary. According to our results, as proven by previous studies, there is a limitation in the expansion potential of hiPSC-ECs when compared with hESC-ECs (Li et al., 2011). Another limitation is cell labeling with DiI, and the use of immunosuppressed mice which require more reliable cell tracking, noninvasive cell tracking, and immunodeficient mice. Furthermore, although autologous hiPSCs minimize (Zhao et al., 2011) or possibly eliminate the need for immune suppression after cell transplantation, however a number of technical issues such as ‘epigenetic memory’ and karyotypic instability during culture (Ji et al., 2010; Kim et al., 2010; Lister et al., 2011; Marchetto et al., 2009; Polo et al., 2010) need to be addressed before they are considered for clinical applications. Some key advances aimed at overcoming these safety concerns have been reached with the production of iPS cells (for review see Gonzalez et al., 2011; Seifinejad et al., 2010; Stadtfeld and Hochedlinger, 2010).