Group were analyzed. Bars = mean 6 SD, ***P,0.001. doi:10.1371/journal.pone.0043643.gNotch Regulates EEPCs and EOCs DifferentiallyFigure 4. RBP-J deficient EEPCs and EOCs display different ability to home into liver during Phx-induced liver regeneration. Normal mice were subjected to PHx. On the day of the operation, mice were transfused order Biotin NHS through the tail 25033180 veins with EEPCs (A, B) or EOCs (C, D) derived from GFP+RBP-J2/2 or GFP+RBP-J+/2 mice. Five days after the transplantation, the livers of the recipient mice were sectioned and stained, and were examined under a fluorescence microscope for GFP+ cells and UEA-1+GFP+ cells (A, C). GFP+ cells and UEA-1+GFP+ cells were quantitatively represented by corresponding pixels (B, D). Bars = mean 6 SD, n = 4, *P,0.05, **P,0.01. doi:10.1371/journal.pone.0043643.gthese cells appear incompetent in directly participating in vessel formation, at least in vitro. In contrast, EOCs could sprout and form vessel-like endothelial cords under appropriate conditions, but EOCs seem not be able to promote liver regeneration in our systems. Moreover, our results suggest that EEPCs and EOCs might take part in liver repair and regeneration through different mechanisms. EEPCs, which express high level of CXCR4, could be recruited to the site of tissue injury by the high level of SDF1a liberated by injured cells [24,25], and participate in tissue repair and regeneration through paracrine factors [42]. EOCs, in contrast, expresses low level of CXCR4, are more destined to ECs and can participate in vessel formation likely through vasculogenesis (Figure S5). Blocking of Notch signaling differentially regulated CXCR4 expression in these two types of cells, likely resulting in their differential homing in the liver. Moreover, these cells might also be chemotracted to the injured tissues mainly by factors other than CXCR4, such as VEGF, which is highly induced by hypoxia through the Hif family transcription factors. Our results showed that the RBP-J-mediated Notch signaling might be critical for the migration and 
function of both EEPCs and EOCs. Notch signaling pathway plays important roles in the colonization, self-renewal, migration and differentiation of EPCs [28]. Our recent study has shown that the Notch signaling pathway might regulate BM-derived EPCs and circulating EPCs differentially, and CXCR4 might play a critical role in these processes. The results reported here, by using in vitro cultured EEPCs and EOCs, are consistent with our previous data and haveconfirmed that Notch signaling plays differential roles in EEPCs and EOCs (Figure S5). EOCs represent more mature EPCs with respect to their lack of expression 
of the precursor cell surface antigens CD34 and CD133. The effect of Notch signaling on EOCs seems more similar to that on ECs, although EOCs can be distinguished from mature ECs by their appearance in in vitro culture and a much higher rate of proliferation [12,43]. In addition to EPCs, Notch signaling also regulates the expression of CXCR4 in other cell types such as mature ECs [44] and dendritic cells [45]. However, the molecular mechanisms by which Notch signaling regulates CXCR4 have not been elucidated yet, leaving the differential regulation of CXCR4 expression in EEPCs and EOCs an open question.Materials and Methods Ethnic statementsThe animal husbandry, experiments and welfare were conducted in NT-157 chemical information accordance with the Detailed Rules for the Administration of Animal Experiments for Medical Research Purpo.Group were analyzed. Bars = mean 6 SD, ***P,0.001. doi:10.1371/journal.pone.0043643.gNotch Regulates EEPCs and EOCs DifferentiallyFigure 4. RBP-J deficient EEPCs and EOCs display different ability to home into liver during Phx-induced liver regeneration. Normal mice were subjected to PHx. On the day of the operation, mice were transfused through the tail 25033180 veins with EEPCs (A, B) or EOCs (C, D) derived from GFP+RBP-J2/2 or GFP+RBP-J+/2 mice. Five days after the transplantation, the livers of the recipient mice were sectioned and stained, and were examined under a fluorescence microscope for GFP+ cells and UEA-1+GFP+ cells (A, C). GFP+ cells and UEA-1+GFP+ cells were quantitatively represented by corresponding pixels (B, D). Bars = mean 6 SD, n = 4, *P,0.05, **P,0.01. doi:10.1371/journal.pone.0043643.gthese cells appear incompetent in directly participating in vessel formation, at least in vitro. In contrast, EOCs could sprout and form vessel-like endothelial cords under appropriate conditions, but EOCs seem not be able to promote liver regeneration in our systems. Moreover, our results suggest that EEPCs and EOCs might take part in liver repair and regeneration through different mechanisms. EEPCs, which express high level of CXCR4, could be recruited to the site of tissue injury by the high level of SDF1a liberated by injured cells [24,25], and participate in tissue repair and regeneration through paracrine factors [42]. EOCs, in contrast, expresses low level of CXCR4, are more destined to ECs and can participate in vessel formation likely through vasculogenesis (Figure S5). Blocking of Notch signaling differentially regulated CXCR4 expression in these two types of cells, likely resulting in their differential homing in the liver. Moreover, these cells might also be chemotracted to the injured tissues mainly by factors other than CXCR4, such as VEGF, which is highly induced by hypoxia through the Hif family transcription factors. Our results showed that the RBP-J-mediated Notch signaling might be critical for the migration and function of both EEPCs and EOCs. Notch signaling pathway plays important roles in the colonization, self-renewal, migration and differentiation of EPCs [28]. Our recent study has shown that the Notch signaling pathway might regulate BM-derived EPCs and circulating EPCs differentially, and CXCR4 might play a critical role in these processes. The results reported here, by using in vitro cultured EEPCs and EOCs, are consistent with our previous data and haveconfirmed that Notch signaling plays differential roles in EEPCs and EOCs (Figure S5). EOCs represent more mature EPCs with respect to their lack of expression of the precursor cell surface antigens CD34 and CD133. The effect of Notch signaling on EOCs seems more similar to that on ECs, although EOCs can be distinguished from mature ECs by their appearance in in vitro culture and a much higher rate of proliferation [12,43]. In addition to EPCs, Notch signaling also regulates the expression of CXCR4 in other cell types such as mature ECs [44] and dendritic cells [45]. However, the molecular mechanisms by which Notch signaling regulates CXCR4 have not been elucidated yet, leaving the differential regulation of CXCR4 expression in EEPCs and EOCs an open question.Materials and Methods Ethnic statementsThe animal husbandry, experiments and welfare were conducted in accordance with the Detailed Rules for the Administration of Animal Experiments for Medical Research Purpo.
