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Plasticity of adult human pancreatic duct cells by neurogenin3-mediated reprogramming.

Swales N, Martens GA, Bonné S, Heremans Y, Borup R, Van de Casteele M, Ling Z, Pipeleers D, Ravassard P, Nielsen F, Ferrer J, Heimberg H - PLoS ONE (2012)

Bottom Line: This neuro-endocrine shift however, is incomplete with less than 10% of full duct-to-endocrine reprogramming achieved.Transduction of exogenous Ngn3 activates endogenous Ngn3 suggesting auto-activation of this gene.Furthermore, pancreatic endocrine reprogramming of human duct cells can be moderately enhanced by inhibition of Delta-Notch signaling as well as by co-expressing the transcription factor Myt1, but not MafA and Pdx1.

View Article: PubMed Central - PubMed

Affiliation: Diabetes Research Center, Vrije Universiteit Brussel, Brussels, Belgium.

ABSTRACT

Aims/hypothesis: Duct cells isolated from adult human pancreas can be reprogrammed to express islet beta cell genes by adenoviral transduction of the developmental transcription factor neurogenin3 (Ngn3). In this study we aimed to fully characterize the extent of this reprogramming and intended to improve it.

Methods: The extent of the Ngn3-mediated duct-to-endocrine cell reprogramming was measured employing genome wide mRNA profiling. By modulation of the Delta-Notch signaling or addition of pancreatic endocrine transcription factors Myt1, MafA and Pdx1 we intended to improve the reprogramming.

Results: Ngn3 stimulates duct cells to express a focused set of genes that are characteristic for islet endocrine cells and/or neural tissues. This neuro-endocrine shift however, is incomplete with less than 10% of full duct-to-endocrine reprogramming achieved. Transduction of exogenous Ngn3 activates endogenous Ngn3 suggesting auto-activation of this gene. Furthermore, pancreatic endocrine reprogramming of human duct cells can be moderately enhanced by inhibition of Delta-Notch signaling as well as by co-expressing the transcription factor Myt1, but not MafA and Pdx1.

Conclusions/interpretation: The results provide further insight into the plasticity of adult human duct cells and suggest measurable routes to enhance Ngn3-mediated in vitro reprogramming protocols for regenerative beta cell therapy in diabetes.

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Related in: MedlinePlus

Modulation of Delta-Notch signaling affects Ngn3 induced endocrine differentiation in adult human duct cells.(A) Real time PCR quantification of endogenous Ngn3, Ins and Syp in AdGFP-Ngn3 transduced duct samples (white bars) versus gamma-secretase inhibitor L6-treated samples (gray bars), shows increased expression levels of endogenous NGN3 (*P<0.05) and the endocrine marker genes (NS, n = 3). Expression of Ngn3, Ins and Syp is reduced when AdGFP-Ngn3 cells are additionally transduced with the Notch1 intracellular domain (NICD) (black bars). Data represent mean ± SEM real-time PCR measurements of target/Ppia mRNA levels compared to AdGFP-Ngn3 transduced duct cells. (B) Conventional PCR showing an increased activation of endogenous HsNgn3 in AdGFP-Ngn3 adult human duct cells treated with L6, but no effect in control transduced duct cells. Controls of exogenous expression (HA-Ngn3 and GFP) as well as expression levels of housekeeping gene GAPDH are shown.
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pone-0037055-g004: Modulation of Delta-Notch signaling affects Ngn3 induced endocrine differentiation in adult human duct cells.(A) Real time PCR quantification of endogenous Ngn3, Ins and Syp in AdGFP-Ngn3 transduced duct samples (white bars) versus gamma-secretase inhibitor L6-treated samples (gray bars), shows increased expression levels of endogenous NGN3 (*P<0.05) and the endocrine marker genes (NS, n = 3). Expression of Ngn3, Ins and Syp is reduced when AdGFP-Ngn3 cells are additionally transduced with the Notch1 intracellular domain (NICD) (black bars). Data represent mean ± SEM real-time PCR measurements of target/Ppia mRNA levels compared to AdGFP-Ngn3 transduced duct cells. (B) Conventional PCR showing an increased activation of endogenous HsNgn3 in AdGFP-Ngn3 adult human duct cells treated with L6, but no effect in control transduced duct cells. Controls of exogenous expression (HA-Ngn3 and GFP) as well as expression levels of housekeeping gene GAPDH are shown.

Mentions: Ngn3 is insufficient for a full reprogramming of duct cells to a genuine endocrine phenotype. Delta-Notch signaling is candidate for permissive instruction of the Ngn3-transduced cells and therefore its role in the reprogramming process was investigated. Exogenous Ngn3 provoked a coordinated activation of genes coding for Delta-Notch signaling proteins, like Dll1, Dll4 [6] and Lnf (Table S1) that could limit reprogramming efficiency. Therefore, the impact of Notch signal inhibition was investigated by loss- and gain-of-function experiments. Addition of the gamma-secretase inhibitor L685,458 (L6) prevents the proteolytic formation of the bioactive intracellular Notch domain (NICD). L6 increased the activation of endogenous HsNgn3 in AdGFP-Ngn3 but had no effect in control transduced duct cells (Figure 4A,B). This accentuation of HsNgn3 expression, however, caused only a tendency towards increased insulin and synaptophysin mRNA levels (Figure 4A). Vice versa, when constitutively active NICD was ectopically expressed in adult human duct cells together with Ngn3, activation of endogenous HsNgn3 was blunted, leading to lower insulin and synaptophysin transcript levels (Figure 4A). Thus, active Notch signaling suppresses Ngn3 autoactivation in adult pancreatic duct cells.


Plasticity of adult human pancreatic duct cells by neurogenin3-mediated reprogramming.

Swales N, Martens GA, Bonné S, Heremans Y, Borup R, Van de Casteele M, Ling Z, Pipeleers D, Ravassard P, Nielsen F, Ferrer J, Heimberg H - PLoS ONE (2012)

Modulation of Delta-Notch signaling affects Ngn3 induced endocrine differentiation in adult human duct cells.(A) Real time PCR quantification of endogenous Ngn3, Ins and Syp in AdGFP-Ngn3 transduced duct samples (white bars) versus gamma-secretase inhibitor L6-treated samples (gray bars), shows increased expression levels of endogenous NGN3 (*P<0.05) and the endocrine marker genes (NS, n = 3). Expression of Ngn3, Ins and Syp is reduced when AdGFP-Ngn3 cells are additionally transduced with the Notch1 intracellular domain (NICD) (black bars). Data represent mean ± SEM real-time PCR measurements of target/Ppia mRNA levels compared to AdGFP-Ngn3 transduced duct cells. (B) Conventional PCR showing an increased activation of endogenous HsNgn3 in AdGFP-Ngn3 adult human duct cells treated with L6, but no effect in control transduced duct cells. Controls of exogenous expression (HA-Ngn3 and GFP) as well as expression levels of housekeeping gene GAPDH are shown.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3351393&req=5

pone-0037055-g004: Modulation of Delta-Notch signaling affects Ngn3 induced endocrine differentiation in adult human duct cells.(A) Real time PCR quantification of endogenous Ngn3, Ins and Syp in AdGFP-Ngn3 transduced duct samples (white bars) versus gamma-secretase inhibitor L6-treated samples (gray bars), shows increased expression levels of endogenous NGN3 (*P<0.05) and the endocrine marker genes (NS, n = 3). Expression of Ngn3, Ins and Syp is reduced when AdGFP-Ngn3 cells are additionally transduced with the Notch1 intracellular domain (NICD) (black bars). Data represent mean ± SEM real-time PCR measurements of target/Ppia mRNA levels compared to AdGFP-Ngn3 transduced duct cells. (B) Conventional PCR showing an increased activation of endogenous HsNgn3 in AdGFP-Ngn3 adult human duct cells treated with L6, but no effect in control transduced duct cells. Controls of exogenous expression (HA-Ngn3 and GFP) as well as expression levels of housekeeping gene GAPDH are shown.
Mentions: Ngn3 is insufficient for a full reprogramming of duct cells to a genuine endocrine phenotype. Delta-Notch signaling is candidate for permissive instruction of the Ngn3-transduced cells and therefore its role in the reprogramming process was investigated. Exogenous Ngn3 provoked a coordinated activation of genes coding for Delta-Notch signaling proteins, like Dll1, Dll4 [6] and Lnf (Table S1) that could limit reprogramming efficiency. Therefore, the impact of Notch signal inhibition was investigated by loss- and gain-of-function experiments. Addition of the gamma-secretase inhibitor L685,458 (L6) prevents the proteolytic formation of the bioactive intracellular Notch domain (NICD). L6 increased the activation of endogenous HsNgn3 in AdGFP-Ngn3 but had no effect in control transduced duct cells (Figure 4A,B). This accentuation of HsNgn3 expression, however, caused only a tendency towards increased insulin and synaptophysin mRNA levels (Figure 4A). Vice versa, when constitutively active NICD was ectopically expressed in adult human duct cells together with Ngn3, activation of endogenous HsNgn3 was blunted, leading to lower insulin and synaptophysin transcript levels (Figure 4A). Thus, active Notch signaling suppresses Ngn3 autoactivation in adult pancreatic duct cells.

Bottom Line: This neuro-endocrine shift however, is incomplete with less than 10% of full duct-to-endocrine reprogramming achieved.Transduction of exogenous Ngn3 activates endogenous Ngn3 suggesting auto-activation of this gene.Furthermore, pancreatic endocrine reprogramming of human duct cells can be moderately enhanced by inhibition of Delta-Notch signaling as well as by co-expressing the transcription factor Myt1, but not MafA and Pdx1.

View Article: PubMed Central - PubMed

Affiliation: Diabetes Research Center, Vrije Universiteit Brussel, Brussels, Belgium.

ABSTRACT

Aims/hypothesis: Duct cells isolated from adult human pancreas can be reprogrammed to express islet beta cell genes by adenoviral transduction of the developmental transcription factor neurogenin3 (Ngn3). In this study we aimed to fully characterize the extent of this reprogramming and intended to improve it.

Methods: The extent of the Ngn3-mediated duct-to-endocrine cell reprogramming was measured employing genome wide mRNA profiling. By modulation of the Delta-Notch signaling or addition of pancreatic endocrine transcription factors Myt1, MafA and Pdx1 we intended to improve the reprogramming.

Results: Ngn3 stimulates duct cells to express a focused set of genes that are characteristic for islet endocrine cells and/or neural tissues. This neuro-endocrine shift however, is incomplete with less than 10% of full duct-to-endocrine reprogramming achieved. Transduction of exogenous Ngn3 activates endogenous Ngn3 suggesting auto-activation of this gene. Furthermore, pancreatic endocrine reprogramming of human duct cells can be moderately enhanced by inhibition of Delta-Notch signaling as well as by co-expressing the transcription factor Myt1, but not MafA and Pdx1.

Conclusions/interpretation: The results provide further insight into the plasticity of adult human duct cells and suggest measurable routes to enhance Ngn3-mediated in vitro reprogramming protocols for regenerative beta cell therapy in diabetes.

Show MeSH
Related in: MedlinePlus