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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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Ngn3 induces its own expression via direct binding on its own promoter.(A) RT-PCR detection of exogenous and endogenous Ngn3 in AdGFP-Ngn3 transduced duct cells. Time course detection of exogenous HA-Ngn3 and endogenous Ngn3 mRNA showing sustained endogenous Ngn3 expression. Time points are indicated in hours (8–64) or days (1–21). Open triangle: genomic Ngn3 PCR fragment; black triangle: endogenous Ngn3 cDNA PCR fragment. Gapdh-specific RT-PCR is included as template control (n = 3). (B) Chromatin immunoprecipitation with an anti-HA antibody on chromatin derived from adult human duct cells transduced with either AdGFP-Ngn3 or AdGFP. DNA from input chromatin was serially diluted as a reference for semiquantitative PCR analysis. In the AdGFP-Ngn3 samples both NGN3 and NEUROD1 promoter fragments are enriched with antibodies directed against the HA tag, suggesting that Ngn3 binds both endogenous wild type promoters. NGN3 and NEUROD1 promoter fragments were not detected in AdGFP control samples, neither were the negative control genomic fragments from the CTLA-1 and BRCA1 genes (n = 2).
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pone-0037055-g003: Ngn3 induces its own expression via direct binding on its own promoter.(A) RT-PCR detection of exogenous and endogenous Ngn3 in AdGFP-Ngn3 transduced duct cells. Time course detection of exogenous HA-Ngn3 and endogenous Ngn3 mRNA showing sustained endogenous Ngn3 expression. Time points are indicated in hours (8–64) or days (1–21). Open triangle: genomic Ngn3 PCR fragment; black triangle: endogenous Ngn3 cDNA PCR fragment. Gapdh-specific RT-PCR is included as template control (n = 3). (B) Chromatin immunoprecipitation with an anti-HA antibody on chromatin derived from adult human duct cells transduced with either AdGFP-Ngn3 or AdGFP. DNA from input chromatin was serially diluted as a reference for semiquantitative PCR analysis. In the AdGFP-Ngn3 samples both NGN3 and NEUROD1 promoter fragments are enriched with antibodies directed against the HA tag, suggesting that Ngn3 binds both endogenous wild type promoters. NGN3 and NEUROD1 promoter fragments were not detected in AdGFP control samples, neither were the negative control genomic fragments from the CTLA-1 and BRCA1 genes (n = 2).

Mentions: Microarray profiling revealed a 12-fold up-regulation of the endogenous human neurogenin3 transcript (HsNgn3) in duct cells 14 days after transduction with cDNA encoding mouse neurogenin3 (MmNgn3) (Table 1). This is surprising since Ngn3 was reported to control its own expression through negative feedback during mouse embryonic development [17]. Because the vector-derived sequence is limited to the open reading frame of MmNgn3 and 11 out of the 12 HsNgn3-specific microarray probes are specific for the 3′ untranslated Ngn3 region, this represents genuine activation of endogenous HsNgn3. Increased expression of HsNgn3 was confirmed by RT-PCR primers that specifically amplify transcripts of either HA-tagged exogenous MmNgn3 cDNA or endogenous HsNgn3 (Figure 3A): the HsNgn3 transcript became detectable 2 days after AdGFP-Ngn3 transduction, peaking at 7 days and then decreased to reach the lower detection limit 3 weeks after transduction. Chromatin immunoprecipitation revealed an in vivo binding of HA-tagged MmNgn3 protein to the 5′ region of the HsNgn3 gene but not to negative control genomic fragments of the CTLA-1 and BRCA1 genes (Figure 3B). These studies therefore provide for the first time evidence for endogenous autoactivation of Ngn3.


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)

Ngn3 induces its own expression via direct binding on its own promoter.(A) RT-PCR detection of exogenous and endogenous Ngn3 in AdGFP-Ngn3 transduced duct cells. Time course detection of exogenous HA-Ngn3 and endogenous Ngn3 mRNA showing sustained endogenous Ngn3 expression. Time points are indicated in hours (8–64) or days (1–21). Open triangle: genomic Ngn3 PCR fragment; black triangle: endogenous Ngn3 cDNA PCR fragment. Gapdh-specific RT-PCR is included as template control (n = 3). (B) Chromatin immunoprecipitation with an anti-HA antibody on chromatin derived from adult human duct cells transduced with either AdGFP-Ngn3 or AdGFP. DNA from input chromatin was serially diluted as a reference for semiquantitative PCR analysis. In the AdGFP-Ngn3 samples both NGN3 and NEUROD1 promoter fragments are enriched with antibodies directed against the HA tag, suggesting that Ngn3 binds both endogenous wild type promoters. NGN3 and NEUROD1 promoter fragments were not detected in AdGFP control samples, neither were the negative control genomic fragments from the CTLA-1 and BRCA1 genes (n = 2).
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Related In: Results  -  Collection

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pone-0037055-g003: Ngn3 induces its own expression via direct binding on its own promoter.(A) RT-PCR detection of exogenous and endogenous Ngn3 in AdGFP-Ngn3 transduced duct cells. Time course detection of exogenous HA-Ngn3 and endogenous Ngn3 mRNA showing sustained endogenous Ngn3 expression. Time points are indicated in hours (8–64) or days (1–21). Open triangle: genomic Ngn3 PCR fragment; black triangle: endogenous Ngn3 cDNA PCR fragment. Gapdh-specific RT-PCR is included as template control (n = 3). (B) Chromatin immunoprecipitation with an anti-HA antibody on chromatin derived from adult human duct cells transduced with either AdGFP-Ngn3 or AdGFP. DNA from input chromatin was serially diluted as a reference for semiquantitative PCR analysis. In the AdGFP-Ngn3 samples both NGN3 and NEUROD1 promoter fragments are enriched with antibodies directed against the HA tag, suggesting that Ngn3 binds both endogenous wild type promoters. NGN3 and NEUROD1 promoter fragments were not detected in AdGFP control samples, neither were the negative control genomic fragments from the CTLA-1 and BRCA1 genes (n = 2).
Mentions: Microarray profiling revealed a 12-fold up-regulation of the endogenous human neurogenin3 transcript (HsNgn3) in duct cells 14 days after transduction with cDNA encoding mouse neurogenin3 (MmNgn3) (Table 1). This is surprising since Ngn3 was reported to control its own expression through negative feedback during mouse embryonic development [17]. Because the vector-derived sequence is limited to the open reading frame of MmNgn3 and 11 out of the 12 HsNgn3-specific microarray probes are specific for the 3′ untranslated Ngn3 region, this represents genuine activation of endogenous HsNgn3. Increased expression of HsNgn3 was confirmed by RT-PCR primers that specifically amplify transcripts of either HA-tagged exogenous MmNgn3 cDNA or endogenous HsNgn3 (Figure 3A): the HsNgn3 transcript became detectable 2 days after AdGFP-Ngn3 transduction, peaking at 7 days and then decreased to reach the lower detection limit 3 weeks after transduction. Chromatin immunoprecipitation revealed an in vivo binding of HA-tagged MmNgn3 protein to the 5′ region of the HsNgn3 gene but not to negative control genomic fragments of the CTLA-1 and BRCA1 genes (Figure 3B). These studies therefore provide for the first time evidence for endogenous autoactivation of Ngn3.

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