Duction [2,14,33,34,47] and sequentially, a new combination of signals involved in exocrine

Duction [2,14,33,34,47] and sequentially, a new combination of signals involved in exocrine development. Among the signalling pathways involved in this cell lineage decision, follistatin stimulates the generation of amylase-expressing cells while repressing the formation of insulin-producing cells [38]. Another signal that may enhance acinar differentiation involves glucocorticoids, which promote acinar differentiation in Pdx1expressing cells at the expense of b-cell proliferation [40]. Moreover, glucocorticoids up-regulate the maturation state of exocrine cells by regulating the expression of amylase, a feature of advanced acinar differentiation and their secretory capability [48]. Therefore, to sustain exocrine differentiation in ESC cultures, cellswere simultaneously treated with both factors as it is unlikely that with one single differentiating agent a robust exocrine differentiation would have been achieved [30]. In agreement with the results of Ren et al. [15], dexamethasone was crucial for an optimal induction of digestive enzyme expression but only when added in combination with the other factors. In this respect, cotreatment with follistatin and FGF7 selectively increased the expression of these markers but to a lesser extent (data not shown). However, the previous combination of factors (Activin A+sodium butyrate+dexamethasone) was somewhat quite inefficient resulting in nearly two-fold increase in the induction of digestive enzymes as compared with control cultures [15]. By contrast, in our experiments there was a substantial increase in the efficiency of induction of digestive enzymes (a factor of 103?04 times as an average estimation) with respect to cultures only treated with 1 SR, which by itself is permissive on ESC acinar differentiation [49]. To further enhance this efficiency, we co-supplemented our cultures with T3, a thyroid hormone that selectively promotes acinar cell proliferation and that cooperates with glucocorticoids in regulating secretory enzyme expression [50,51]. With addition of T3 we did not observe a significant impact on the magnitude of acinar gene expression (data not shown). Additional detailed studies will be needed to determine the role of individual differentiating factors in our new method presented here; however, a recent study showed that FGF7 is able to regulate acinar differentiation in mESC [14]. Nonetheless, this protocol was also useful for endocrine differentiation, thus missing a more specific cell lineage approach. In this regard, we previously showed that FGF7 induced the expression of both endocrine and exocrine markers in mESC, supporting its role in the expansion rather the differentiation of pancreatic or lineage progenitors [30,41]. Therefore, the search for more selective SMER-28 combinations of molecules for the generation of exocrine cells remains necessary. In this sense, a valuable contribution of our protocol is that it favours exocrine differentiation over the endocrine phenotype. It is likely that the differentiation agents used herein impinge on the early endocrine commitment of pancreatic progenitors as suggested by a significant decrease of Ngn3 PHCCC biological activity message levels (Fig. 4). Notably, Nkx6.1 was also down-regulated in line with recent data showing a mutually antagonist action with Ptf1a in directing endocrine versus acinar cell fate choices [52]. In keeping with this, a significant reduction in the number of cells expressing Ins and Gluc was observed (Fig. 5). Remarkably, the maj.Duction [2,14,33,34,47] and sequentially, a new combination of signals involved in exocrine development. Among the signalling pathways involved in this cell lineage decision, follistatin stimulates the generation of amylase-expressing cells while repressing the formation of insulin-producing cells [38]. Another signal that may enhance acinar differentiation involves glucocorticoids, which promote acinar differentiation in Pdx1expressing cells at the expense of b-cell proliferation [40]. Moreover, glucocorticoids up-regulate the maturation state of exocrine cells by regulating the expression of amylase, a feature of advanced acinar differentiation and their secretory capability [48]. Therefore, to sustain exocrine differentiation in ESC cultures, cellswere simultaneously treated with both factors as it is unlikely that with one single differentiating agent a robust exocrine differentiation would have been achieved [30]. In agreement with the results of Ren et al. [15], dexamethasone was crucial for an optimal induction of digestive enzyme expression but only when added in combination with the other factors. In this respect, cotreatment with follistatin and FGF7 selectively increased the expression of these markers but to a lesser extent (data not shown). However, the previous combination of factors (Activin A+sodium butyrate+dexamethasone) was somewhat quite inefficient resulting in nearly two-fold increase in the induction of digestive enzymes as compared with control cultures [15]. By contrast, in our experiments there was a substantial increase in the efficiency of induction of digestive enzymes (a factor of 103?04 times as an average estimation) with respect to cultures only treated with 1 SR, which by itself is permissive on ESC acinar differentiation [49]. To further enhance this efficiency, we co-supplemented our cultures with T3, a thyroid hormone that selectively promotes acinar cell proliferation and that cooperates with glucocorticoids in regulating secretory enzyme expression [50,51]. With addition of T3 we did not observe a significant impact on the magnitude of acinar gene expression (data not shown). Additional detailed studies will be needed to determine the role of individual differentiating factors in our new method presented here; however, a recent study showed that FGF7 is able to regulate acinar differentiation in mESC [14]. Nonetheless, this protocol was also useful for endocrine differentiation, thus missing a more specific cell lineage approach. In this regard, we previously showed that FGF7 induced the expression of both endocrine and exocrine markers in mESC, supporting its role in the expansion rather the differentiation of pancreatic or lineage progenitors [30,41]. Therefore, the search for more selective combinations of molecules for the generation of exocrine cells remains necessary. In this sense, a valuable contribution of our protocol is that it favours exocrine differentiation over the endocrine phenotype. It is likely that the differentiation agents used herein impinge on the early endocrine commitment of pancreatic progenitors as suggested by a significant decrease of Ngn3 message levels (Fig. 4). Notably, Nkx6.1 was also down-regulated in line with recent data showing a mutually antagonist action with Ptf1a in directing endocrine versus acinar cell fate choices [52]. In keeping with this, a significant reduction in the number of cells expressing Ins and Gluc was observed (Fig. 5). Remarkably, the maj.