Only a portion of the day 7 colonies kept growing and could be detected selleckchem as large colonies at days 14 and 21, whereas the majority of colonies stopped expanding and
disappeared by days 14 and 21 (Fig. 1B). Although the total number of large colonies did not differ significantly between wild-type and Bmi1−/− Dlk+ cells at day 7 of culture (Fig. 1B), the diameter of colonies derived from Bmi1−/− Dlk+ cells was slightly reduced (Fig. 1C). The impeded expansion of Bmi1−/− Dlk+ cell-derived colonies was obvious at day 14 of culture (Fig. 1B,C). Approximately 10% of large colonies from wild-type Dlk+ cells continued to proliferate up to day 21 of culture, whereas no colonies derived from Bmi1−/− Dlk+ cells expanded beyond day 21 (Fig. 1B). It has been reported that Dlk+ cells are composed of albumin (Alb)+ cytokeratin 7 (CK7)+ cells and Alb+CK7−
cells, and Alb+CK7+ cells mainly contribute to the regeneration I-BET-762 in vitro in retrorsine-treated liver.16, 17 These findings suggest that Alb+CK7+ cells, which have the capacity to give rise to both Alb+CK7− and Alb−Ck7+ progenies, function as hepatic stem/progenitor cells. Therefore, the quantification of Alb+CK7+ impotent cells is one of the approaches to evaluate the content of hepatic stem/progenitor cells, although not all Alb+CK7+ cells necessarily have the capacity for bipotential differentiation. Immunocytochemical analyses revealed that the ability of Bmi1−/− Dlk+ cells to differentiate into Alb+ hepatocytes and CK7+ cholangiocytes was preserved (Fig. 1D). However, the absolute number of Alb+CK7+ bipotent cells were significantly decreased in large colonies derived from Bmi1−/− Dlk+ cells compared to those in wild-type large colonies (Fig. 1D,E). The absolute number of Alb+CK7+ cells per each large colony was 7.6 ± 1.5 and 2.8 ± 0.4, respectively (P < 0.05) (Fig.
1E). Consistent with these findings, flow cytometric analyses demonstrated that the Dlk+ population in Bmi1−/− colonies decreased rapidly compared to that in wild-type colonies (Fig. 1F). The Dlk+ fraction in wild-type colonies was 1.1% ± 0.2% at day 7 and 0.7% ± 0.1% at day 14 of culture, whereas that in Bmi1−/− colonies was 0.5% ± 0.1% and 0.3% ± 0.1%, respectively. medchemexpress Conversely, forced expression of Bmi1 in wild-type Dlk+ cells significantly promoted colony expansion (Supporting Fig. 2A-C) and increased the Dlk+ fraction and number of bipotent cells (Supporting Fig. 2D,E). Oval cells, although their origin is controversial, have been considered stem/progenitor cells in adult liver.18 Histological analyses demonstrated a drastic decrease in A6-positive oval cell numbers in 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-treated Bmi1−/− adult liver (Supporting Fig. 3). Together, these findings suggest that Bmi1 plays an important role in the maintenance and expansion of stem/progenitor cells in both fetal and adult livers.
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