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, 10 (10), e0140352
eCollection

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

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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

Kenneth R Muir et al. PLoS One.

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.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Islet enriched pancreatic cells form fibroblast-like monolayers and dedifferentiate in adherent cell culture.
A: Phase contrast images taken in culture from day 0 to passage 6. QRT/PCR analysis of endocrine, epithelial, mesenchymal (B) and pluripotency markers (C) in cells harvested from passage 1 to passage 9 in tissue culture. Data are expressed relative to glyceraldehyde 3-phosphate dehydrogenase. ESC are human embryonic stem cells.
Fig 2
Fig 2. KLF4 overexpression induces morphological change with up-regulation of epithelial markers and down-regulation of mesenchymal markers.
Islet enriched pancreatic cell clusters were cultured in RPMI with 10% FBS and allowed to adhere and expand. (A) At passage 6 cells were transduced with Ad-Klf4. The left panel shows untreated cells (NA) and the right panel cells treated with Ad-KLf4. Cells were stained with DAPI (nuclei) and anti-KLF4 (green). (B) QRT/PCR analysis of exogenous (mouse) Klf4 expression up to day 10 in transduced (klf4) and untreated (NA) cells. The dotted line represents the time of adenoviral transduction. Data are expressed relative to glyceraldehyde 3-phosphate dehydrogenase and represent mean ± SEM (n = 3). (C) Phase-contrast images showing morphological changes at time points post infection with Ad-Klf4 or Ad-EGFP. (D) Cells were harvested at time points for gene expression by QRT/PCR. Data were expressed relative to glyceraldehyde-3-phosphate dehydrogenase (n = 3). A two-way ANOVA was performed with Bonferroni post hoc test comparing treatment groups with Ad-EGFP. For all analyses, *P < 0.05 **P < 0.01 ***P < 0.001. (E) Immunocytochemical staining of the epithelial marker E-cadherin and the mesenchymal marker vimentin at day 4 post transduction with Ad-Klf4 versus control. Nuclei were counterstained with DAPI. Scale bar = 20μm.
Fig 3
Fig 3. KLF4 overexpression induces re-expression of both endocrine and exocrine markers.
Islet enriched pancreatic cell clusters were cultured in RPMI with 10% FBS and allowed to adhere and expand. At passage 6 cells were transduced with Ad-Klf4 or Ad-EGFP. (A) QRT/PCR analysis of pancreatic markers in transduced cells. The dotted line represents the time of adenoviral transduction. Data represent mean ± SEM (n = 3) (B) QRT/PCR analysis of insulin (INS) and glucagon (GCG) expression in cells transduced with Ad-Klf4 or Ad-Klf4 in combination with Ad-Pdx1, Ad-Ngn3, Ad-MafA and Ad-Pax4 (4TFs). Data are expressed relative to glyceraldehyde 3-phosphate dehydrogenase and expressed as mean ± SEM (n = 3). Samples transduced with Ad-Klf4 or Ad-EGFP were immunostained at day 4 for E-cadherin, C-peptide and Chromogranin A (C) and for Amylase and CK19 (D). Nuclei were counterstained with DAPI. Scale bar = 20μm.
Fig 4
Fig 4. Effect of KLF4 is transient and induces apoptosis in treated cells.
Islet-derived MSCs at passage 6 were transduced with lenti-KLF4 or lenti-EGFP and harvested at time points. (A) QRT/PCR analysis for expression of the indicated genes following culture for the indicated days after transduction with Lenti-Klf4 or Lenti-EGFP. The data are expressed relative to glyceraldehyde-3-phosphate dehydrogenase and represent the mean ± SEM (n = 3). A two-way ANOVA was performed on all QRT/PCR analyses with Bonferroni post hoc test comparing treatment groups with lenti-GFP. For all analyses, P *< 0.05 **P < 0.01 ***P < 0.001. (B) Cleaved caspase 3 (CASP3) co-expresses with E-cadherin (ECAD) in Ad-Klf4 but not Ad-EGFP treated cells. Nuclei were counterstained with DAPI. Scale bar = 20μm. (C) Cells were subjected to UV light or transduced with Ad-KLF4 or Ad-EGFP (NA) and fixed at day 4. A TUNEL assay was performed followed by counterstaining with DAPI. Over 1500 nuclei were counted per treatment and cells identified as apoptotic calculated as a percentage of all cells.
Fig 5
Fig 5. Suspension culture enhances the effects of Ad-Klf4.
Islet-derived MSCs at passage 6 were transduced with Ad-Klf4 or Ad-EGFP and cultured overnight in adherent cell culture conditions. The cells were then either left in adherent conditions or transferred to suspension culture for a further 4 days. (A) Phase contrast comparison of cells in adherent or suspension culture at day 5. (B) Samples were harvested for QRT/PCR at several time points and analysed for expression of pancreatic, epithelial and mesenchymal markers. Data were expressed relative to glyceraldehyde-3-phosphate dehydrogenase and represent the mean ± SEM (n = 3). A one-way ANOVA was performed on all QRT/PCR analyses with Dunnett post hoc test comparing treatment groups with an Ad-EGFP control. Unpaired t-tests were performed where necessary. For all analyses, P *< 0.05 **P < 0.01 ***P < 0.001.
Fig 6
Fig 6. β-cell derived MSCs can be differentiated towards adipocyte and osteoblast lineages.
(A) Islets were transduced 24h after plating with a lenti virus encoding Cre recombinase under the control of the insulin promoter (Le-INS-Cre) and a lentivirus encoding dsRED preceded by a floxed stop cassette (Le-dsRED). These lineage traced beta cells were allowed to expand in culture for 6 weeks. (B) The expanded dsRed+ beta-derived MSCs were FACS sorted and cultured up to passage 4. Left panel dsRed, middle DAPI, and right merged. (C) Immunostaining of sorted β-cell derived MSCs upon differentiation towards adipocytes (lipid droplets, LipidTox) or osteoblasts (osteocalcin). Scale bar = 50μm. Quantification of the percentage of differentiation towards adipocytes (D) and osteoblasts (E). >1500 nuclei were counted per treatment and cells identified as positive calculated as a percentage of total cells.
Fig 7
Fig 7. Ad-Klf4 induces INS-dsRed MSCs to differentiate down both endocrine and exocrine lineages.
(A) FACS sorted and expanded INS-dsRED MSCs were transduced with Ad-Klf4, samples fixed for immunocytochemistry and stained for E-cadherin, chromogranin A and CK19. Nuclei were counterstained with DAPI. Scale bar = 20μm. (B) FACS sorted and expanded INS-dsRED MSCs were transduced with Ad-Klf4 or Ad-EGFP. Samples were harvested for QRT/PCR analysed for expression of epithelial and endocrine markers, relative to glyceraldehyde-3-phosphate dehydrogenase and represented as mean ± SEM (n = 3). Unpaired t-tests were performed between Ad-Klf4 and Ad-EGFP transduced cells. For all analyses, P *< 0.05 **P < 0.01 ***P < 0.001.

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