Krüppel-Like Factor 4 Overexpression Initiates a Mesenchymal-to-Epithelial Transition and Redifferentiation of Human Pancreatic Cells following Expansion in Long Term Adherent Culture

Kenneth R Muir, Maria João Lima, Hilary M Docherty, Neil W A McGowan, Shareen Forbes, Yves Heremans, Stuart J Forbes, Harry Heimberg, John Casey, Kevin Docherty

Research output: Contribution to journalArticle

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Abstract

A replenishable source of insulin-producing cells has the potential to cure type 1 diabetes. Attempts to culture and expand pancreatic β-cells in vitro have resulted in their transition from insulin-producing epithelial cells to mesenchymal stromal cells (MSCs) with high proliferative capacity but devoid of any hormone production. The aim of this study was to determine whether the transcription factor Krüppel-like factor 4 (KLF4), could induce a mesenchymal-to-epithelial transition (MET) of the cultured cells. Islet-enriched pancreatic cells, allowed to dedifferentiate and expand in adherent cell culture, were transduced with an adenovirus containing KLF4 (Ad-Klf4). Cells were subsequently analysed for changes in cell morphology by light microscopy, and for the presence of epithelial and pancreatic markers by immunocytochemistry and quantitative RT/PCR. Infection with Ad-Klf4 resulted in morphological changes, down-regulation of mesenchymal markers, and re-expression of both epithelial and pancreatic cell markers including insulin and transcription factors specific to β-cells. This effect was further enhanced by culturing cells in suspension. However, the effects of Ad-KLf4 were transient and this was shown to be due to increased apoptosis in Klf4-expressing cells. Klf4 has been recently identified as a pioneer factor with the ability to modulate the structure of chromatin and enhance reprogramming/transdifferentiation. Our results show that Klf4 may have a role in the redifferentiation of expanded pancreatic cells in culture, but before this can be achieved the off-target effects that result in increased apoptosis would need to be overcome.

Original languageEnglish
Article numbere0140352
Number of pages16
JournalPloS ONE
Volume10
Issue number10
Early online date12 Oct 2015
DOIs
Publication statusPublished - 12 Oct 2015

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Epithelial-Mesenchymal Transition
Insulin
Transcription Factors
Apoptosis
cells
Medical problems
Cell culture
Chromatin
Optical microscopy
insulin
Suspensions
Adenoviridae
Cells
Hormones
cell culture
apoptosis
transcription factors
Cell Culture Techniques
Epithelial Cells
Adenoviridae Infections

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Krüppel-Like Factor 4 Overexpression Initiates a Mesenchymal-to-Epithelial Transition and Redifferentiation of Human Pancreatic Cells following Expansion in Long Term Adherent Culture. / Muir, Kenneth R; Lima, Maria João; Docherty, Hilary M; McGowan, Neil W A; Forbes, Shareen; Heremans, Yves; Forbes, Stuart J; Heimberg, Harry; Casey, John; Docherty, Kevin.

In: PloS ONE, Vol. 10, No. 10, e0140352, 12.10.2015.

Research output: Contribution to journalArticle

Muir, KR, Lima, MJ, Docherty, HM, McGowan, NWA, Forbes, S, Heremans, Y, Forbes, SJ, Heimberg, H, Casey, J & Docherty, K 2015, 'Krüppel-Like Factor 4 Overexpression Initiates a Mesenchymal-to-Epithelial Transition and Redifferentiation of Human Pancreatic Cells following Expansion in Long Term Adherent Culture', PloS ONE, vol. 10, no. 10, e0140352. https://doi.org/10.1371/journal.pone.0140352
Muir, Kenneth R ; Lima, Maria João ; Docherty, Hilary M ; McGowan, Neil W A ; Forbes, Shareen ; Heremans, Yves ; Forbes, Stuart J ; Heimberg, Harry ; Casey, John ; Docherty, Kevin. / Krüppel-Like Factor 4 Overexpression Initiates a Mesenchymal-to-Epithelial Transition and Redifferentiation of Human Pancreatic Cells following Expansion in Long Term Adherent Culture. In: PloS ONE. 2015 ; Vol. 10, No. 10.
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abstract = "A replenishable source of insulin-producing cells has the potential to cure type 1 diabetes. Attempts to culture and expand pancreatic β-cells in vitro have resulted in their transition from insulin-producing epithelial cells to mesenchymal stromal cells (MSCs) with high proliferative capacity but devoid of any hormone production. The aim of this study was to determine whether the transcription factor Kr{\"u}ppel-like factor 4 (KLF4), could induce a mesenchymal-to-epithelial transition (MET) of the cultured cells. Islet-enriched pancreatic cells, allowed to dedifferentiate and expand in adherent cell culture, were transduced with an adenovirus containing KLF4 (Ad-Klf4). Cells were subsequently analysed for changes in cell morphology by light microscopy, and for the presence of epithelial and pancreatic markers by immunocytochemistry and quantitative RT/PCR. Infection with Ad-Klf4 resulted in morphological changes, down-regulation of mesenchymal markers, and re-expression of both epithelial and pancreatic cell markers including insulin and transcription factors specific to β-cells. This effect was further enhanced by culturing cells in suspension. However, the effects of Ad-KLf4 were transient and this was shown to be due to increased apoptosis in Klf4-expressing cells. Klf4 has been recently identified as a pioneer factor with the ability to modulate the structure of chromatin and enhance reprogramming/transdifferentiation. Our results show that Klf4 may have a role in the redifferentiation of expanded pancreatic cells in culture, but before this can be achieved the off-target effects that result in increased apoptosis would need to be overcome.",
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