Robert P. Fordham’s research while affiliated with University of Cambridge and other places

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Publications (7)


Document S1. Figures S1–S6 and Tables S1 and S2
  • Data
  • File available

January 2018

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11 Reads

Shiro Yui

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Luca Azzolin

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Martti Maimets

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[...]

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Kim B. Jensen
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Supplementary Material 2

January 2018

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5 Reads

Table S3. Ranked Gene List from the Comparison of Fetal Signature Genes with the Expression Data from Ulcerative Colitis Patients and Normal Individuals, Related to Figure 2


Figure 1. Repairing Intestinal Epithelium Has a Cellular and Molecular Profile Distinct from the Normal Epithelium (A) Macroscopic images of the distal part of the colon in an untreated mouse (left) and a mouse 2 weeks post-administration of DSS (right). The demarcated area indicates regions undergoing active re-epithelialization. Scale bar, 2 mm. (B) H&E staining of homeostatic tissue and tissue in the repair phase at 2 weeks following DSS administration. Scale bar, 100 mm. (C) The mucosal/submucosal thickness at homeostasis and repair phase (2 weeks). Shown are mean distances ± SEM (n = 4 animals; p = 0.007 based on twosided Student's t test). (D) Sca1 (green) expression in colonic epithelium in homeostatic and repair phase. Sections are counterstained with DAPI (blue). The demarcated line indicates the epithelial structure. Scale bar, 100 mm.
Figure 3. The Repairing Epithelium Displays Features of Active Mechano-Transduction (A) Differentially expressed probes ranked in heatmap comparing tissue homeostasis and repair phase (n = 3; 2-fold regulated; FDR < 0.05). Selected genes associated with lineage differentiation and extracellular matrix interactions are indicated. (legend continued on next page)
Figure 4. Rebuilding the Repairing Epithelium In Vitro Based on Defined Components (A) Murine small intestinal epithelial cells cultured in the presence of EGF, Noggin, and R-spondin1 (ENR) or with the addition of Wnt3a (+Wnt3a) in either Matrigel or collagen type I. Scale bars, 100 mm. (B) Heatmap of differentially expressed probe sets between culture conditions using MG/ENR+Wnt3a (ENRW, green) and COL/ENRW (red) (n = 6; fold change > 1.5; FDR < 0.1). Examples of differentially expressed genes are indicated. (C) Detection of F-actin with Phalloidin (gray) counterstained with DAPI (blue) and YAP (green) counterstained with E-cadherin (red) and DAPI (blue) in Matrigel and collagen type I cultures from the small intestine. Insets show enlarged view of the indicated regions. Scale bars, 50 mm in the top panels and 100 mm in the bottom panels. (D) Heatmap of Z score-transformed relative expression levels as determined by qPCR for selected YAP/TAZ target genes and markers of the fetal and adult states in cultures of small intestinal epithelial cells grown in either Matrigel (MG) or collagen type I (Col) in the presence of EGF/Noggin/Rspondin (ENR) with or without Wnt3a (W). (legend continued on next page)
Figure 5. YAP/TAZ Transcriptional Activation Is Required for Cellular Reprogramming (A) Normal organoids (WT) and Apc-knockout (Apc KO ) spheres derived from the small intestine cultured in collagen type I with the indicated cytokine cocktail. Time point of analysis is indicated in the left bar. Scale bar, 100 mm. (legend continued on next page)
Figure 6. Injury-Induced Cellular Reprogramming Is Reversible (A and B) Serial sections of engrafted patches from Matrigel cultures analyzed for (A) tdTomato (red) and Sca1 (green) and (B) YAP (green) and E-cadherin (red) 1 day after transplantation (day 12). Scale bar, 100 mm. (C) Diagram of the transplantation strategy using cells from the conditional YAP/TAZ cdKO cells and control animals. Indicated are the administration of DSS (day 0-5), time points for transplantation (days 8 and 11), administration of 4-hydroxy tamoxifen (days 12 and 13), as well as the final analysis (day 16). (D) Whole-mount analysis of the colon for control (tdTomato+/red) and Villin CreER YAP/TAZ cDKO cells (eGFP+/green) before (day 12) and after (day 16) tamoxifen administration. Arrows illustrate areas of GFP+ cells. (E) Quantification of the ratio of the area covered by GFP-versus RFP-expressing cells before and after tamoxifen treatment. Each dot represents independent animals, and data are presented as the mean ± SEM (p = 0.029 based on a Mann-Whitney exact one-sided test). (legend continued on next page)
YAP/TAZ-Dependent Reprogramming of Colonic Epithelium Links ECM Remodeling to Tissue Regeneration

December 2017

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562 Reads

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494 Citations

Cell Stem Cell

Tissue regeneration requires dynamic cellular adaptation to the wound environment. It is currently unclear how this is orchestrated at the cellular level and how cell fate is affected by severe tissue damage. Here we dissect cell fate transitions during colonic regeneration in a mouse dextran sulfate sodium (DSS) colitis model, and we demonstrate that the epithelium is transiently reprogrammed into a primitive state. This is characterized by de novo expression of fetal markers as well as suppression of markers for adult stem and differentiated cells. The fate change is orchestrated by remodeling the extracellular matrix (ECM), increased FAK/Src signaling, and ultimately YAP/TAZ activation. In a defined cell culture system recapitulating the extracellular matrix remodeling observed in vivo, we show that a collagen 3D matrix supplemented with Wnt ligands is sufficient to sustain endogenous YAP/TAZ and induce conversion of cell fate. This provides a simple model for tissue regeneration, implicating cellular reprogramming as an essential element.


Generation of Multipotent Foregut Stem Cells from Human Pluripotent Stem Cells

October 2013

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289 Reads

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89 Citations

Stem Cell Reports

Human pluripotent stem cells (hPSCs) could provide an infinite source of clinically relevant cells with potential applications in regenerative medicine. However, hPSC lines vary in their capacity to generate specialized cells, and the development of universal protocols for the production of tissue-specific cells remains a major challenge. Here, we have addressed this limitation for the endodermal lineage by developing a defined culture system to expand and differentiate human foregut stem cells (hFSCs) derived from hPSCs. hFSCs can self-renew while maintaining their capacity to differentiate into pancreatic and hepatic cells. Furthermore, near-homogenous populations of hFSCs can be obtained from hPSC lines which are normally refractory to endodermal differentiation. Therefore, hFSCs provide a unique approach to bypass variability between pluripotent lines in order to obtain a sustainable source of multipotent endoderm stem cells for basic studies and to produce a diversity of endodermal derivatives with a clinical value.


Figure 1. Derivation of Immature Intestinal Progenitors from Human Fetal and Pluripotent Cells (A) Whole mount of human gestational week 10 small intestine. (B) Higher magnification of villi (arrow) and intervillus regions (arrowhead) in (A). (C-E) Immunohistochemistry analysis for Ki67 (C), PAS staining (D), and Lysozyme (E) in week 10 human small intestine. (F and G) Spheroid cultures from week 10 human small intestinal epithelium, grown with (G) and without (F) prostaglandin E2 (PGE2) (2.5 mM). (H and I) Intestinal tissue derived from directed differentiation of human induced pluripotent stem cells (hiPSCs), cultured with (I) and without (H) PGE2. (J) Relative expression levels of intestinal lineage markers in material from undifferentiated human induced pluripotent stem cells (hiPSC), iPSCderived intestine (Int. diff.), human primary fetal enterospheres (hFEnS), human adult organoids (hOrgs), primary fetal human small intestine (FhSI), and primary adult human small intestine (AhSI). Red and green colors reflect increased and decreased deviation from the mean, respectively. (K) Detection of VILLIN (green) and CHGA (red) in hiPSC-FEnS. The scale bars represent 2 mm in (A) and 100 mm in (C)-(E) and (K). See also Figure S1 and Table S1.
Figure 2. Establishment of mFEnS from Immature Mouse Intestine (A and B) Immunohistochemistry analysis for Phospho-Histone-H3 (pHist) on sections of small intestine from E16 mice (A) and P15 mice (B). (C) Relative expression levels of intestinal lineage markers in tissue isolated from proximal murine intestine at increasing developmental age from E16 to adult. Red and green colors reflect increased and decreased deviation from the mean, respectively. (D–H) Representative images of in vitro structures derived from E14 to P15. The arrow and arrowhead in (G) indicate an FEnS and an organoid, respectively. (I) Relative proportions of FEnS and organoids present after 2 weeks from E16, P2, and P15 tissues. (J) Metaphase spread of a cell at day 180 shows a normal karyotype (n = 15). (K and L) Detection of apical villin expression (green) in adult small intestine (K) and mFEnS (L). (M–P) Lysozyme expression in adult small intestine (M), cross sections of mFEnS (N), and whole-mount organoids and mFEnS (O and P). (Q and R) BrdU incorporation analysis in whole mounts of organoids and FEnS (green). b -catenin (red) is used as a counterstain. The scale bars represent 100 m m. E, embryonic day; P, postnatal day; adult, >3 weeks postnatal. See also Figures S2 and S3. 
Figure 3. Adult Stem Cell Behavior Follows a Caudal to Rostral Pattern (A) Schematic diagram of the Proximal, Mid, and Distal parts of the small intestine and the representative images of cultures derived at P2. (B) Relative proportion of FEnS and organoids in the different sections of the small intestine. (C) Expression analysis in material isolated from Proximal, Mid, and Distal regions. Data represent the mean, and the error bars, the SEM (n = 3). Data are expressed relative to Proximal, on a Log 2 scale. 
Figure 4. In Vitro Maturation of Fetal Enteric Progenitors Is Associated with Lgr5 Expression and Wnt Signaling (A) Detection of Lgr5-EGFP at P2 from Lgr5-EGFP-ires-CreERT2 mice. (B) Isolation of Lgr5-EGFP +ve and Lgr5-EGFP À ve epithelial cells from P2 small intestine by flow cytometry. 
Transplantation of Expanded Fetal Intestinal Progenitors Contributes to Colon Regeneration after Injury

October 2013

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233 Reads

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369 Citations

Cell Stem Cell

Regeneration and homeostasis in the adult intestinal epithelium is driven by proliferative resident stem cells, whose functional properties during organismal development are largely unknown. Here, we show that human and mouse fetal intestine contains proliferative, immature progenitors, which can be expanded in vitro as Fetal Enterospheres (FEnS). A highly similar progenitor population can be established during intestinal differentiation of human induced pluripotent stem cells. Established cultures of mouse fetal intestinal progenitors express lower levels of Lgr5 than mature progenitors and propagate in the presence of the Wnt antagonist Dkk1, and new cultures can be induced to form mature intestinal organoids by exposure to Wnt3a. Following transplantation in a colonic injury model, FEnS contribute to regeneration of colonic epithelium by forming epithelial crypt-like structures expressing region-specific differentiation markers. This work provides insight into mechanisms underlying development of the mammalian intestine and points to future opportunities for patient-specific regeneration of the digestive tract.


Reporting Live from the Epidermal Stem Cell Compartment!

August 2012

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9 Reads

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2 Citations

Cell Stem Cell

Hair follicle regeneration is controlled by an intricate relationship between epidermal stem cells and their microenvironment. A recent report in Nature by Rompolas et al. (2012) uses two-photon live imaging to interrogate the spatial organization and cellular requirements for hair follicle regeneration by epidermal stem cells and their immediate progeny.

Citations (4)


... Accumulating evidence indicates that inflammation in the intestine leads to uncontrolled ECM remodeling and alters the mechanical properties. Yui et al. demonstrated that DSS-induced colitis leads to significant ECM remodeling in a YAP/TAZ signalingdependent manner [118]. ECM remodeling is a key feature of IBD and is associated with disease progression and inflammation. ...

Reference:

Mechanobiological Approach for Intestinal Mucosal Immunology
YAP/TAZ-Dependent Reprogramming of Colonic Epithelium Links ECM Remodeling to Tissue Regeneration

Cell Stem Cell

... The Hippo signaling pathway is essential for regulating pancreatic development, as well as β cell proliferation, differentiation and survival. Overexpression of YAP-S6A in pancreatic progenitor cells reduces the differentiation efficiency into β-like cells, but increases the number of proliferating β-like cells [26,93,[99][100][101][102]. FGF10, fibroblast growth factor 10; EGF, epidermal growth factor; TGFβ, transforming growth factor β; Inh, inhibitor; P, passage; Ha, harmine; LY, LY364947; LIF, leukemia inhibitory factor; DE, definitive endoderm; FG, foregut; PP, pancreatic progenitor; TesR1, mTESR™1; BMP4, bone morphogenetic protein 4; bFGF, basic fibroblast growth factor; VEGF, vascular endothelial growth factor; MEF, mouse embryonic fibroblast; HGF, hepatocyte growth factor; OE, overexpression. ...

Generation of Multipotent Foregut Stem Cells from Human Pluripotent Stem Cells

Stem Cell Reports

... For example, researchers have demonstrated that mouse colon organoids can be effectively expanded in vitro and successfully transplanted into damaged mice 145 , where they remodel functional crypt units. Similarly, fetal progenitor-derived small intestinal organoids have exhibited the ability to integrate and function in vivo 146 . When intestinal organoids derived from PSCs were implanted under the kidney capsule of mice, they displayed good permeability and peptide uptake, highlighting their potential in treating conditions like short bowel syndrome 147 . ...

Transplantation of Expanded Fetal Intestinal Progenitors Contributes to Colon Regeneration after Injury

Cell Stem Cell

... Skin is the largest organ of the human body; it plays a key role in protecting the body against pathogens. The homeostasis of skin tissue is maintained by rare but pluripotent epidermal stem cells (EpSCs) and their progeny, transient amplifying (TA) cells [1,2]. As pluripotent cells, EpSCs not only have an unlimited self-renewal capability to maintain a certain population but they also differentiate to form structures such as hair follicles and sebaceous glands [2,3]. ...

Reporting Live from the Epidermal Stem Cell Compartment!
  • Citing Article
  • August 2012

Cell Stem Cell