Targeted Mutation of NGN3 Gene Disrupts Pancreatic Endocrine Cell Development in Pigs

Sheets, Timothy P.; Park, Ki‐Eun; Park, Chi-Hun; Swift, Steven M.; Powell, Anne M.; Donovan, David M.; Telugu, Bhanu P.

doi:10.1038/s41598-018-22050-0

Cited by 32 publications

(26 citation statements)

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“…While a previous study has demonstrated that culturing young porcine islets for 20 days in a multistep differentiation media involving the use of 6 different agents, including oncostatin M, dexamethasone, nicotinamide, exendin‐4, TGF‐ß1, and thrombin, resulted in a 2‐fold increase in the composition of beta and alpha cells, Nec‐1 treatment alone in our study increased the proportion of beta, alpha, and delta cells by 2‐fold on both days 3 and 7 of culture . The transcription factor Ngn3 directs the differentiation of pancreatic precursor cells toward the development of endocrine cells, but is suppressed after final differentiation of endocrine cells . In accordance with the increase in the composition of mature endocrine cells with prolonged culture in both control and untreated islets, the level of Ngn3‐positive endocrine progenitor cells decreased with culture time in our study, indicating the development of mature endocrine cells from progenitor cells.…”

Section: Discussioncontrasting

confidence: 40%

Necrostatin‐1 supplementation enhances young porcine islet maturation and in vitro function

Lau

Corrales

Alexander

et al. 2019

Xenotransplantation

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Background Necroptosis has been demonstrated to be a primary mechanism of islet cell death. This study evaluated whether the supplementation of necrostatin‐1 (Nec‐1), a potent inhibitor of necroptosis, to islet culture media could improve the recovery, maturation, and function of pre‐weaned porcine islets (PPIs). Methods PPIs were isolated from pre‐weaned Yorkshire piglets (8‐15 days old) and either cultured in control islet culture media (n = 6) or supplemented with Nec‐1 (100 µM, n = 5). On days 3 and 7 of culture, islets were assessed for recovery, insulin content, viability, cellular composition, GLUT2 expression in beta cells, differentiation of pancreatic endocrine progenitor cells, function, and oxygen consumption rate. Results Nec‐1 supplementation induced a 2‐fold increase in the insulin content of PPIs on day 7 of culture. When compared to untreated islets, Nec‐1 treatment doubled the beta‐ and alpha‐cell composition and accelerated the development of delta cells. Additionally, beta cells of Nec‐1‐treated islets had a significant upregulation in GLUT2 expression. The enhanced development of major endocrine cells and GLUT2 expression after Nec‐1 treatment subsequently led to a significant increase in the amount of insulin secreted in response to in vitro glucose challenge. Islet recovery, viability, and oxygen consumption rate were unaffected by Nec‐1. Conclusion This study underlines the importance of necroptosis in islet cell death after isolation and demonstrates the novel effects of Nec‐1 to increase islet insulin content, enhance pancreatic endocrine cell development, facilitate GLUT2 upregulation in beta cells, and augment insulin secretion. Nec‐1 supplementation to culture media significantly improves islet quality prior to xenotransplantation.

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Section: Discussioncontrasting

confidence: 40%

Necrostatin‐1 supplementation enhances young porcine islet maturation and in vitro function

Lau

Corrales

Alexander

et al. 2019

Xenotransplantation

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“…In an effort to test the utility of pXEN cells as nuclear donors, we performed SCNT with the pXEN cells used in the chimera assay (above), alongside previously published crossbred knock-out fetal fibroblasts (FF NGN3-/-) as controls (25). A total of 222 cloned embryos reconstituted from pXEN (Xnt pCOL1A:GFP #3-2, n=61) and FF (n=161) were co-transferred into two surrogate gilts to exclude confounding variables associated with recipient animals affecting the outcome.…”

Section: Generation Of Viable Cloned Offspring From Pxen Cells Via Scntmentioning

confidence: 99%

“…$ data obtained from our previous study. *NGN3 -/cells originated from our previous report25 . All the fetal fibroblasts and pXEN cells with the exception of NGN3 -/cells used as SCNT donors were derived from the same fetus (female Ossabow fetal fibroblast #6).…”

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Chimera-competent eXtra-Embryonic eNdoderm (XEN) cells established from pig embryos

Park

Jeoung

et al. 2020

Preprint

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Classification Biological Sciences/ Agricultural Sciences Keywords primitive endoderm, stem cell, chimera, XEN, SCNT Author Contributions CP, JCB and BT conceived the project and designed the experiments; CP, YJ, YU and JB established and performed characterization of embryonic outgrowths and XEN cells; KP and AP performed the knockin and generated the GFP reporter lines used for generating GFP:XEN cells used in chiera experiments; CP, JL, LP, TL, JS, YG, YS, JW 2 generated the libraries and transcriptomic analysis; CP and BT wrote the initial draft, CP, JW, JCB and BT revised the manuscript based on the input from all authors. All authors approved final draft for submission. Abstract In this article, we report for the first time the derivation and characterization of extraembryonic endoderm (XEN) cells from primitive endoderm (PrE) of porcine (p) embryos. The pXEN cells can be reliably and reproducibly generated from parthenote, in vitro and in vivo derived embryos. The pXEN cells retained all the hallmarks of PrE including expression of canonical PrE and XEN cell markers (GATA4, GATA6, SOX17, SALL4, FOXA2, and HNF4A). Transcriptome analysis further confirmed their XEN cell origin. The pXEN cells when introduced into blastocyst stage embryo contributed to widespread chimerism including visceral yolk sac, chorion, as well as embryonic gut and liver primordium in the fetus. The pXEN cells were shown to be an efficient nuclear donor for generating cloned offspring. Taken together, pXEN cells fulfil a longstanding need for a stable, chimera-competent, and nuclear transfer-compatible porcine embryonic cells with applications for agriculture and medicine. Significance StatementWe report for the first time, the derivation and characterization of extraembryonic endoderm (XEN) stem cells from porcine (p) embryos. The pXEN cells can be reliably and reproducibly derived from primitive endoderm precursors. When injected into blastocyst-stage embryos, the pXEN cells have contributed to wide-spread chimerism including visceral yolk sac, chorion of the extraembryonic membranes, as well as definitive endoderm of the fetus, primarily the embryonic gut and liver primordium.Additionally, these XEN cells have proven to be an efficient nuclear donor for generating cloned offspring. These newly discovered stem cells provide a novel model for studying 4 lineage segregation, as well as a source for interspecies chimeras for generating endodermal organs, and for genome editing in livestock. 5

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“…Humans and pigs are omnivorous mammals with similar physiology and metabolic diseases, including diabetes mellitus and its complications, from diet-induced obesity, insulinopathy and bcell stress [9][10][11] . Recent experimental advances expand possible studies of pig pancreas development to include gain and loss-of-function genetics [12][13][14][15] , fetal surgery and immunomodulation 16 , and primary islet cell genetics 17 . Prior studies of pig pancreas development have largely relied on immunohistological survey of tissue cell types [18][19][20][21] .…”

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confidence: 99%

Molecular and genetic regulation of pig pancreatic islet cell development

Kim

Whitener

Peiris

et al. 2019

Preprint

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Reliance on rodents for understanding pancreatic genetics, developmental biology and islet function could limit progress in developing interventions for human diseases like diabetes mellitus.Similarities of pancreas morphology and function, and pancreatic disease modeling in pigs suggest that porcine and human pancreas developmental biology may have useful homologies.However, little is known about pig pancreas development. To fill this knowledge gap, we investigated fetal and neonatal pig pancreas at multiple, crucial developmental stages using modern experimental approaches. Purification of islet β-, α-and d-cells via flow cytometry followed by high-throughput transcriptome analysis (RNA-Seq) provided comprehensive gene expression profiles. Morphometric analysis revealed dynamic developmental endocrine cell allocation and islet architectural similarities between pig and human islets. Gene expression analysis identified cell-and stage-specific expression in sorted pig β-and α-cells, and revealed that pig and human β-cells and α-cells shared characteristic molecular and developmental features not observed in mouse β-cells or α-cells. Over 150 causal or candidate 'diabetes risk' genes identified by human studies had dynamic developmental expression in pig β-and α-cells. Our analysis also unveiled scores of signaling pathways linked to native islet β-cell functional maturation. Thus, the findings, conceptual advances, and resources detailed here show how pig pancreatic islet studies complement or surpass other systems for understanding the developmental programs that generate functional β-and a-cells, and that are relevant to human diseases like diabetes mellitus.

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Targeted Mutation of NGN3 Gene Disrupts Pancreatic Endocrine Cell Development in Pigs

Cited by 32 publications

References 50 publications

Necrostatin‐1 supplementation enhances young porcine islet maturation and in vitro function

Necrostatin‐1 supplementation enhances young porcine islet maturation and in vitro function

Chimera-competent eXtra-Embryonic eNdoderm (XEN) cells established from pig embryos

Molecular and genetic regulation of pig pancreatic islet cell development

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