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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

Overexpression of pancreatic endocrine transcription factors in adult human duct cells.(A, B) Effect of Myt1b overexpression in adult human duct cells. Adult human duct cells have been transduced with AdGFP, AdGFP-Ngn3 and/or AdGFP-Myt1 with a total MOI 100 and gene expression profiles have been analyzed 10 dpt. (A) Increased levels of several neuroendocrine genes are observed in AdGFP-Myt1 duct cells. The effects are enhanced by overexpression of Myt1 and Ngn3. (B) Except for Pax4, Myt1 does not upregulate known Ngn3 target genes. P<0.05 vs AdGFP (*) or AdGFP-Myt1 (#); P<0.005 vs AdGFP (**) or AdGFP-Myt1 (##); P<0.001 vs AdGFP (***) or AdGFP-Myt1 (###). (C, D) Adenoviral overexpression of Pdx1, Ngn3 and MafA in adult human duct cells does not induce the neuroendocrine genes Ins and Syp. Adult human duct cells have been transduced with control viruses AdGFP and AdNull, AdGFP-Ngn3+AdGFP+AdNull or AdGFP-Pdx1+AdGFP-Ngn3+AdMafA with a total MOI of 100 and gene expression has been analyzed 10 dpt. RT-qPCR analysis reveals that none of these conditions induced insulin or synaptophysin transcripts. Data represent mean ± SEM real-time PCR measurements of target/Ppia mRNA levels compared to non-transduced adult human duct cells (n ≥3).
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pone-0037055-g005: Overexpression of pancreatic endocrine transcription factors in adult human duct cells.(A, B) Effect of Myt1b overexpression in adult human duct cells. Adult human duct cells have been transduced with AdGFP, AdGFP-Ngn3 and/or AdGFP-Myt1 with a total MOI 100 and gene expression profiles have been analyzed 10 dpt. (A) Increased levels of several neuroendocrine genes are observed in AdGFP-Myt1 duct cells. The effects are enhanced by overexpression of Myt1 and Ngn3. (B) Except for Pax4, Myt1 does not upregulate known Ngn3 target genes. P<0.05 vs AdGFP (*) or AdGFP-Myt1 (#); P<0.005 vs AdGFP (**) or AdGFP-Myt1 (##); P<0.001 vs AdGFP (***) or AdGFP-Myt1 (###). (C, D) Adenoviral overexpression of Pdx1, Ngn3 and MafA in adult human duct cells does not induce the neuroendocrine genes Ins and Syp. Adult human duct cells have been transduced with control viruses AdGFP and AdNull, AdGFP-Ngn3+AdGFP+AdNull or AdGFP-Pdx1+AdGFP-Ngn3+AdMafA with a total MOI of 100 and gene expression has been analyzed 10 dpt. RT-qPCR analysis reveals that none of these conditions induced insulin or synaptophysin transcripts. Data represent mean ± SEM real-time PCR measurements of target/Ppia mRNA levels compared to non-transduced adult human duct cells (n ≥3).

Mentions: Myelin transcription factor 1 (Myt1) is another important islet cell transcription factor and works in concert with Ngn3 to promote endocrine cell differentiation in the developing pancreas [11], [12], [13]. Myt1-encoding mRNA was absent from normal duct cells and although ectopic Ngn3 activated Myt1 gene expression we over-expressed Myt1b, the Myt1 isoform that is predominantly active in embryonic chicken and mouse pancreas [11], [12], to investigate whether more Myt1, alone or in combination with Ngn3 could enhance duct-to-endocrine cell reprogramming. After 10 days, Myt1b alone enhanced transcript levels of the insulin and glucagon genes to a minor extent and of the synaptophysin gene to a major extent. Only the latter one and glucokinase were increased in duct cells transduced by Ngn3 only (Figure 5A). Introduction of Myt1b in Ngn3-expressing duct cells further increased transcript levels of synaptophysin and glucokinase (P<0.05) but not of insulin and glucagon (Figure 5A). However, no significant increase in protein levels of insulin and synaptophysin could be detected. The Ngn3-induced neuro-endocrine shift occurs through recapitulation of embryonic neuro-endocrine differentiation [6], i.e. by activation of its direct target genes [6], [18], [19], [20], [21]. The abundance of transcripts coding for NeuroD1, Pax4 and Insm1 increased in duct cells transduced with Ngn3 but not Myt1 only. Myt1b further enhanced the amount of Ngn3-induced Pax4 mRNA (4-fold, P<0.001) (Figure 5B). The lack of additive effects of Myt1b on most direct targets of Ngn3 suggests that Myt1b and Ngn3 can act through parallel pathways.


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)

Overexpression of pancreatic endocrine transcription factors in adult human duct cells.(A, B) Effect of Myt1b overexpression in adult human duct cells. Adult human duct cells have been transduced with AdGFP, AdGFP-Ngn3 and/or AdGFP-Myt1 with a total MOI 100 and gene expression profiles have been analyzed 10 dpt. (A) Increased levels of several neuroendocrine genes are observed in AdGFP-Myt1 duct cells. The effects are enhanced by overexpression of Myt1 and Ngn3. (B) Except for Pax4, Myt1 does not upregulate known Ngn3 target genes. P<0.05 vs AdGFP (*) or AdGFP-Myt1 (#); P<0.005 vs AdGFP (**) or AdGFP-Myt1 (##); P<0.001 vs AdGFP (***) or AdGFP-Myt1 (###). (C, D) Adenoviral overexpression of Pdx1, Ngn3 and MafA in adult human duct cells does not induce the neuroendocrine genes Ins and Syp. Adult human duct cells have been transduced with control viruses AdGFP and AdNull, AdGFP-Ngn3+AdGFP+AdNull or AdGFP-Pdx1+AdGFP-Ngn3+AdMafA with a total MOI of 100 and gene expression has been analyzed 10 dpt. RT-qPCR analysis reveals that none of these conditions induced insulin or synaptophysin transcripts. Data represent mean ± SEM real-time PCR measurements of target/Ppia mRNA levels compared to non-transduced adult human duct cells (n ≥3).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3351393&req=5

pone-0037055-g005: Overexpression of pancreatic endocrine transcription factors in adult human duct cells.(A, B) Effect of Myt1b overexpression in adult human duct cells. Adult human duct cells have been transduced with AdGFP, AdGFP-Ngn3 and/or AdGFP-Myt1 with a total MOI 100 and gene expression profiles have been analyzed 10 dpt. (A) Increased levels of several neuroendocrine genes are observed in AdGFP-Myt1 duct cells. The effects are enhanced by overexpression of Myt1 and Ngn3. (B) Except for Pax4, Myt1 does not upregulate known Ngn3 target genes. P<0.05 vs AdGFP (*) or AdGFP-Myt1 (#); P<0.005 vs AdGFP (**) or AdGFP-Myt1 (##); P<0.001 vs AdGFP (***) or AdGFP-Myt1 (###). (C, D) Adenoviral overexpression of Pdx1, Ngn3 and MafA in adult human duct cells does not induce the neuroendocrine genes Ins and Syp. Adult human duct cells have been transduced with control viruses AdGFP and AdNull, AdGFP-Ngn3+AdGFP+AdNull or AdGFP-Pdx1+AdGFP-Ngn3+AdMafA with a total MOI of 100 and gene expression has been analyzed 10 dpt. RT-qPCR analysis reveals that none of these conditions induced insulin or synaptophysin transcripts. Data represent mean ± SEM real-time PCR measurements of target/Ppia mRNA levels compared to non-transduced adult human duct cells (n ≥3).
Mentions: Myelin transcription factor 1 (Myt1) is another important islet cell transcription factor and works in concert with Ngn3 to promote endocrine cell differentiation in the developing pancreas [11], [12], [13]. Myt1-encoding mRNA was absent from normal duct cells and although ectopic Ngn3 activated Myt1 gene expression we over-expressed Myt1b, the Myt1 isoform that is predominantly active in embryonic chicken and mouse pancreas [11], [12], to investigate whether more Myt1, alone or in combination with Ngn3 could enhance duct-to-endocrine cell reprogramming. After 10 days, Myt1b alone enhanced transcript levels of the insulin and glucagon genes to a minor extent and of the synaptophysin gene to a major extent. Only the latter one and glucokinase were increased in duct cells transduced by Ngn3 only (Figure 5A). Introduction of Myt1b in Ngn3-expressing duct cells further increased transcript levels of synaptophysin and glucokinase (P<0.05) but not of insulin and glucagon (Figure 5A). However, no significant increase in protein levels of insulin and synaptophysin could be detected. The Ngn3-induced neuro-endocrine shift occurs through recapitulation of embryonic neuro-endocrine differentiation [6], i.e. by activation of its direct target genes [6], [18], [19], [20], [21]. The abundance of transcripts coding for NeuroD1, Pax4 and Insm1 increased in duct cells transduced with Ngn3 but not Myt1 only. Myt1b further enhanced the amount of Ngn3-induced Pax4 mRNA (4-fold, P<0.001) (Figure 5B). The lack of additive effects of Myt1b on most direct targets of Ngn3 suggests that Myt1b and Ngn3 can act through parallel pathways.

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