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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

Extent of Ngn3-mediated duct-to-islet endocrine reprogramming.Transcripts that are ≥1.5-fold (LB) higher expressed in untransduced human endocrine versus suspension cultured duct cells (n = 1128 on the HG133A array, panel A, green) were compared to the 140 transcripts that were AdGFP-Ngn3-activated as compared to AdGFP-transduced duct cells using the same statistical criteria (panel A and B, gray). Nearly half (45%, 63 of 140) of Ngn3-regulated transcripts (panel A, intersect) are expressed in endocrine cells. Panel B compares these 140 Ngn3-activated transcripts (gray) to a previously reported [16] panel of 332 genes with beta cell-selective expression in mouse, rat and human. Ngn3 activated 24 of these endocrine marker genes. Gene chip mRNA expression signal (mean±SD, n = 3) in transduced duct cells is shown in panels C and D.
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pone-0037055-g002: Extent of Ngn3-mediated duct-to-islet endocrine reprogramming.Transcripts that are ≥1.5-fold (LB) higher expressed in untransduced human endocrine versus suspension cultured duct cells (n = 1128 on the HG133A array, panel A, green) were compared to the 140 transcripts that were AdGFP-Ngn3-activated as compared to AdGFP-transduced duct cells using the same statistical criteria (panel A and B, gray). Nearly half (45%, 63 of 140) of Ngn3-regulated transcripts (panel A, intersect) are expressed in endocrine cells. Panel B compares these 140 Ngn3-activated transcripts (gray) to a previously reported [16] panel of 332 genes with beta cell-selective expression in mouse, rat and human. Ngn3 activated 24 of these endocrine marker genes. Gene chip mRNA expression signal (mean±SD, n = 3) in transduced duct cells is shown in panels C and D.

Mentions: Adult human duct cells were transduced with adenoviruses expressing Ngn3 and GFP (AdGFP-Ngn3) or GFP only (AdGFP) and cultured in suspension for 3 or 14 days to study their genome-wide mRNA expression. Tables S1 and S2 list all transcripts that were either 1.5-fold (LB) up- or down regulated in Ngn3-transduced duct cells at day 3 (68 up, 36 down), day 14 (105 up, 19 down) or both. The Ngn3-associated transcriptome shift consisted of 188 different genes that were altered at least at one of the 2 time points tested (140 up and 48 down). Ngn3-activated genes were however statistically (P<0.001) enriched in gene ontologies relating to development, neurogenesis, synaptic transmission, regulated hormone secretion and nuclear regulators. Genes belonging to these overrepresented clusters are shown in Table 1. Conventional and real-time PCR confirmed activation of genes coding for beta cell transcription factors (NeuroD1, Pax4, Nkx2.2), vesicle proteins (Scg3, Syp) insulin processing (Pcsk1) and nutrient sensing (Gck), but also genes that are considered characteristic for neural cells but are present in beta cells as well (Pcp4, Rab26) (Figure S1).


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)

Extent of Ngn3-mediated duct-to-islet endocrine reprogramming.Transcripts that are ≥1.5-fold (LB) higher expressed in untransduced human endocrine versus suspension cultured duct cells (n = 1128 on the HG133A array, panel A, green) were compared to the 140 transcripts that were AdGFP-Ngn3-activated as compared to AdGFP-transduced duct cells using the same statistical criteria (panel A and B, gray). Nearly half (45%, 63 of 140) of Ngn3-regulated transcripts (panel A, intersect) are expressed in endocrine cells. Panel B compares these 140 Ngn3-activated transcripts (gray) to a previously reported [16] panel of 332 genes with beta cell-selective expression in mouse, rat and human. Ngn3 activated 24 of these endocrine marker genes. Gene chip mRNA expression signal (mean±SD, n = 3) in transduced duct cells is shown in panels C and D.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3351393&req=5

pone-0037055-g002: Extent of Ngn3-mediated duct-to-islet endocrine reprogramming.Transcripts that are ≥1.5-fold (LB) higher expressed in untransduced human endocrine versus suspension cultured duct cells (n = 1128 on the HG133A array, panel A, green) were compared to the 140 transcripts that were AdGFP-Ngn3-activated as compared to AdGFP-transduced duct cells using the same statistical criteria (panel A and B, gray). Nearly half (45%, 63 of 140) of Ngn3-regulated transcripts (panel A, intersect) are expressed in endocrine cells. Panel B compares these 140 Ngn3-activated transcripts (gray) to a previously reported [16] panel of 332 genes with beta cell-selective expression in mouse, rat and human. Ngn3 activated 24 of these endocrine marker genes. Gene chip mRNA expression signal (mean±SD, n = 3) in transduced duct cells is shown in panels C and D.
Mentions: Adult human duct cells were transduced with adenoviruses expressing Ngn3 and GFP (AdGFP-Ngn3) or GFP only (AdGFP) and cultured in suspension for 3 or 14 days to study their genome-wide mRNA expression. Tables S1 and S2 list all transcripts that were either 1.5-fold (LB) up- or down regulated in Ngn3-transduced duct cells at day 3 (68 up, 36 down), day 14 (105 up, 19 down) or both. The Ngn3-associated transcriptome shift consisted of 188 different genes that were altered at least at one of the 2 time points tested (140 up and 48 down). Ngn3-activated genes were however statistically (P<0.001) enriched in gene ontologies relating to development, neurogenesis, synaptic transmission, regulated hormone secretion and nuclear regulators. Genes belonging to these overrepresented clusters are shown in Table 1. Conventional and real-time PCR confirmed activation of genes coding for beta cell transcription factors (NeuroD1, Pax4, Nkx2.2), vesicle proteins (Scg3, Syp) insulin processing (Pcsk1) and nutrient sensing (Gck), but also genes that are considered characteristic for neural cells but are present in beta cells as well (Pcp4, Rab26) (Figure S1).

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