Functional Genomic Screening in Human Pluripotent Stem Cells Reveals New Roadblocks in Early Pancreatic Endoderm Formation

Krüger, Jana; Breunig, Markus; Pasquini, L; Morawe, Mareen; Groß, Alexander; Arnold, Frank; Russell, Ronan; Seufferlein, Thomas; Azoitei, Ninel; Kestler, Hans A.; Julier, Cécile; Heller, Sandra; Hohwieler, Meike; Kleger, Alexander

doi:10.3390/cells11030582

Cited by 3 publications

(5 citation statements)

References 56 publications

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“…Finally, we wanted to challenge the relevance of TBX3 in human pancreatic development. Thus, we employed an inducible knockdown of TBX3 by an shRNA in a previously reported iPSC line [ 12 ] during in vitro pancreatic progenitor differentiation [ 58 – 61 ]. We assessed trilineage potential (generation of acinar, ductal, and endocrine cells) in a recently described porcine urinary bladder (PUB) organ culture model for the maturation of pancreatic progenitor cells [ 62 , 63 ] (Additional file 1 : Fig.…”

Section: Resultsmentioning

confidence: 99%

TBX3 is dynamically expressed in pancreatic organogenesis and fine-tunes regeneration

et al. 2023

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Background The reactivation of genetic programs from early development is a common mechanism for injury-induced organ regeneration. T-box 3 (TBX3) is a member of the T-box family of transcription factors previously shown to regulate pluripotency and subsequent lineage commitment in a number of tissues, including limb and lung. TBX3 is also involved in lung and heart organogenesis. Here, we provide a comprehensive and thorough characterization of TBX3 and its role during pancreatic organogenesis and regeneration. Results We interrogated the level and cell specificity of TBX3 in the developing and adult pancreas at mRNA and protein levels at multiple developmental stages in mouse and human pancreas. We employed conditional mutagenesis to determine its role in murine pancreatic development and in regeneration after the induction of acute pancreatitis. We found that Tbx3 is dynamically expressed in the pancreatic mesenchyme and epithelium. While Tbx3 is expressed in the developing pancreas, its absence is likely compensated by other factors after ablation from either the mesenchymal or epithelial compartments. In an adult model of acute pancreatitis, we found that a lack of Tbx3 resulted in increased proliferation and fibrosis as well as an enhanced inflammatory gene programs, indicating that Tbx3 has a role in tissue homeostasis and regeneration. Conclusions TBX3 demonstrates dynamic expression patterns in the pancreas. Although TBX3 is dispensable for proper pancreatic development, its absence leads to altered organ regeneration after induction of acute pancreatitis.

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Section: Resultsmentioning

confidence: 99%

TBX3 is dynamically expressed in pancreatic organogenesis and fine-tunes regeneration

et al. 2023

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“…Cell-cell adhesion molecules such as Cadherin-1 and -2 were predominantly found in the progenitor populations (Figure S11D). Interestingly, the ligand DSC2, recently published as a relevant signaling cue for endoderm formation [72], was mainly expressed in ductal-like progenitors 3, transmitting signals to progenitor 1-3 and endocrine clusters via the DSG2 receptor (Figure S11E). Proliferative cues mostly originated from progenitor cluster 3 which was exemplified by selected ligand-receptor pairs (GAS6-TYRO3, MDK-NCL) (Figure S11F) [73].…”

Section: Distinct Ligand-receptor Interactions Specify the Progenitor...mentioning

confidence: 99%

Single-cell profiling of GP2-enriched pancreatic progenitors to simultaneously create acinar, ductal, and endocrine organoids

Merz¹,

Breunig²,

Melzer³

et al. 2023

Theranostics

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Rationale: Pancreatic lineage specification follows the formation of tripotent pancreatic progenitors (PPs). Current protocols rebuilding PPs in vitro have an endocrine lineage bias and are mostly based on PDX1/NKX6-1 coexpression neglecting other markers decisive for PP heterogeneity and lineage potential. However, true tripotent PPs are of utmost interest to study also exocrine disorders such as pancreatic cancer and to simultaneously generate all three pancreatic lineages from the same ancestor. Methods: Here, we performed a comprehensive compound testing to advance the generation of multipotent progenitors, which were further characterized for their trilineage potential in vitro and in vivo . The heterogeneity and cell-cell communication across the PP subpopulations were analyzed via single-cell transcriptomics. Results: We introduce a novel PP differentiation platform based on a comprehensive compound screening with an advanced design of experiments computing tool to reduce impurities and to increase Glycoprotein-2 expression and subsequent trilineage potential. Superior PP tripotency was proven in vitro by the generation of acinar, endocrine, and ductal cells as well as in vivo upon orthotopic transplantation revealing all three lineages at fetal maturation level. GP2 expression levels at PP stage ascribed varying pancreatic lineage potential. Intermediate and high GP2 levels were superior in generating endocrine and duct-like organoids (PDLO). FACS-based purification of the GP2 high PPs allowed the generation of pancreatic acinar-like organoids (PALO) with proper morphology and expression of digestive enzymes. scRNA-seq confirmed multipotent identity, positioned the GP2/PDX1/NKX6-1 high population next to human fetal tip and trunk progenitors and identified novel ligand-receptor (LR) interactions in distinct PP subpopulations. LR validation experiments licensed midkine and VEGF signaling to increase markers labelling the single cell clusters with high GP2 expression. Conclusion: In this study, we guide human pluripotent stem cells into multipotent pancreatic progenitors. This common precursor population, which has the ability to mature into acinar, ductal and functional β-cells, serves as a basis for studying developmental processes and deciphering early cancer formation in a cell type-specific context. Using single-cell RNA sequencing and subsequent validation studies, we were able to dissect PP heterogeneity and specific cell-cell communication signals.

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“…Cells were analysed for relevant differentiation markers at the human embryonic stem cell (hESC), DE, PE, and PDLO stages. Surface marker staining was performed with living cells, as previously described [27,30], using the following antibodies: CXCR4 (PE-conjugated; 1:50; MHCXCR404, Life Technologies, Carlsbad, CA, USA), c-Kit (APC-conjugated; 1:100, CD11705, Thermo Fisher Scientific), TRA1-60 (FITC-conjugated; 1:10; 560380, BD Biosciences, Franklin Lakes, NJ, USA), and SSEA4 (PE-conjugated; 1:10; 560128; BD Biosciences).…”

Section: Flow Cytometrymentioning

confidence: 99%

“…At the PE and PP stages, intracellular staining of transcription factors PDX1 and NKX6-1 was performed. Cells were fixed in 4% paraformaldehyde and 100 mM sucrose for 25 min on ice and stained as described [27,29,30] using anti-NKX6-1 (1:150, F55A12, DSHB, Iowa City, IA, USA), anti-PDX1 (1:250, AF2419, R&D Systems, Minneapolis, MN, USA), and donkey-anti-mouse and donkey-anti-goat (Alexa Fluor 488-, 568-, or 647-conjugated, 1:500, Thermo Fisher Scientific). For live measurement of the mCherry reporter signal during monolayer culture, cells were harvested with TrypLE and resuspended in 2% FCS/PBS.…”

Section: Flow Cytometrymentioning

confidence: 99%

“…High levels of KRT19 have also been documented in patient serum [22] or plasma [23] and generally correlate with worse patient survival [24] and metastatic progression [25]. Albeit several studies have investigated KRT19 in non-pancreatic cancer types [14]; in the pancreas it is mostly employed as a diagnostic marker [24], leaving unexplored its role during pancreatic development and cancer [26][27][28].…”

Section: Introductionmentioning

confidence: 99%

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DNA methylation‐associated allelic inactivation regulates Keratin 19 gene expression during pancreatic development and carcinogenesis

Krüger

Fischer

Breunig

et al. 2023

The Journal of Pathology

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Within the pancreas, Keratin 19 (KRT19) labels the ductal lineage and is a determinant of pancreatic ductal adenocarcinoma (PDAC). To investigate KRT19 expression dynamics, we developed a human pluripotent stem cell (PSC)‐based KRT19‐mCherry reporter system in different genetic backgrounds to monitor KRT19 expression from its endogenous gene locus. A differentiation protocol to generate mature pancreatic duct‐like organoids was applied. While KRT19/mCherry expression became evident at the early endoderm stage, mCherry signal was present in nearly all cells at the pancreatic endoderm (PE) and pancreatic progenitor (PP) stages. Interestingly, despite homogenous KRT19 expression, mCherry positivity dropped to 50% after ductal maturation, indicating a permanent switch from biallelic to monoallelic expression. DNA methylation profiling separated the distinct differentiation intermediates, with site‐specific DNA methylation patterns occurring at the KRT19 locus during ductal maturation. Accordingly, the monoallelic switch was partially reverted upon treatment with a DNA‐methyltransferase inhibitor. In human PDAC cohorts, high KRT19 levels correlate with low locus methylation and decreased survival. At the same time, activation of oncogenic KRASG12D signalling in our reporter system reversed monoallelic back to biallelic KRT19 expression in pancreatic duct‐like organoids. Allelic reactivation was also detected in single‐cell transcriptomes of human PDACs, which further revealed a positive correlation between KRT19 and KRAS expression. Accordingly, KRAS mutant PDACs had higher KRT19 mRNA but lower KRT19 gene locus DNA methylation than wildtype counterparts. KRT19 protein was additionally detected in plasma of PDAC patients, with higher concentrations correlating with shorter progression‐free survival in gemcitabine/nabPaclitaxel‐treated and opposing trends in FOLFIRINOX‐treated patients. Apart from being an important pancreatic ductal lineage marker, KRT19 appears tightly controlled via a switch from biallelic to monoallelic expression during ductal lineage entry and is aberrantly expressed after oncogenic KRASG12D expression, indicating a role in PDAC development and malignancy. Soluble KRT19 might serve as a relevant biomarker to stratify treatment. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

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Functional Genomic Screening in Human Pluripotent Stem Cells Reveals New Roadblocks in Early Pancreatic Endoderm Formation

Cited by 3 publications

References 56 publications

TBX3 is dynamically expressed in pancreatic organogenesis and fine-tunes regeneration

TBX3 is dynamically expressed in pancreatic organogenesis and fine-tunes regeneration

Single-cell profiling of GP2-enriched pancreatic progenitors to simultaneously create acinar, ductal, and endocrine organoids

DNA methylation‐associated allelic inactivation regulates Keratin 19 gene expression during pancreatic development and carcinogenesis

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