Engineering of three-dimensional microenvironments to promote contractile behavior in primary intestinal organoids.
2014; 6 (2): 127-142
Interfollicular Epidermal Stem Cells Self-Renew via Autocrine Wnt Signaling
2013; 342 (6163): 1226-1230
Multiple culture techniques now exist for the long-term maintenance of neonatal primary murine intestinal organoids in vitro; however, the achievement of contractile behavior within cultured organoids has thus far been infrequent and unpredictable. Here we combine finite element simulation of oxygen transport and quantitative comparative analysis of cellular microenvironments to elucidate the critical variables that promote reproducible intestinal organoid contraction. Experimentally, oxygen distribution was manipulated by adjusting the ambient oxygen concentration along with the use of semi-permeable membranes to enhance transport. The culture microenvironment was further tailored through variation of collagen type-I matrix density, addition of exogenous R-spondin1, and specification of culture geometry. "Air-liquid interface" cultures resulted in significantly higher numbers of contractile cultures relative to traditional submerged cultures. These interface cultures were confirmed to have enhanced and more symmetric oxygen transport relative to traditional submerged cultures. While oxygen availability was found to impact in vitro contraction rate and the orientation of contractile movement, it was not a key factor in enabling contractility. For all conditions tested, reproducible contractile behavior only occurred within a consistent and narrow range of collagen type-I matrix densities with porosities of approximately 20% and storage moduli near 30 Pa. This suggests that matrix density acts as a "permissive switch" that enables contractions to occur. Similarly, contractions were only observed in cultures with diameters less than 15.5 mm that had relatively large interfacial surface area between the compliant matrix and the rigid culture dish. Taken together, these data suggest that spatial geometry and mechanics of the microenvironment, which includes both the encapsulating matrix as well as the surrounding culture device, may be key determinants of intestinal organoid functionality. As peristaltic contractility is a crucial requirement for normal digestive tract function, this achievement of reproducible organoid contraction marks a pivotal advancement towards engineering physiologically functional replacement tissue constructs.
View details for DOI 10.1039/c3ib40188j
View details for PubMedID 24343706
A multicenter study to standardize reporting and analyses of fluorescence-activated cell-sorted murine intestinal epithelial cells
AMERICAN JOURNAL OF PHYSIOLOGY-GASTROINTESTINAL AND LIVER PHYSIOLOGY
2013; 305 (8): G542-G551
The skin is a classical example of a tissue maintained by stem cells. However, the identity of the stem cells that maintain the interfollicular epidermis and the source of the signals that control their activity remain unclear. Using mouse lineage tracing and quantitative clonal analyses, we showed that the Wnt target gene Axin2 marks interfollicular epidermal stem cells. These Axin2-expressing cells constitute the majority of the basal epidermal layer, compete neutrally, and require Wnt/β-catenin signaling to proliferate. The same cells contribute robustly to wound healing, with no requirement for a quiescent stem cell subpopulation. By means of double-labeling RNA in situ hybridization in mice, we showed that the Axin2-expressing cells themselves produce Wnt signals as well as long-range secreted Wnt inhibitors, suggesting an autocrine mechanism of stem cell self-renewal.
View details for DOI 10.1126/science.1239730
View details for Web of Science ID 000327857900046
View details for PubMedID 24311688
Restriction of intestinal stem cell expansion and the regenerative response by YAP
2013; 493 (7430): 106-?
Fluorescence-activated cell sorting (FACS) is an essential tool for studies requiring isolation of distinct intestinal epithelial cell populations. Inconsistent or lack of reporting of the critical parameters associated with FACS methodologies has complicated interpretation, comparison, and reproduction of important findings. To address this problem a comprehensive multicenter study was designed to develop guidelines that limit experimental and data reporting variability and provide a foundation for accurate comparison of data between studies. Common methodologies and data reporting protocols for tissue dissociation, cell yield, cell viability, FACS, and postsort purity were established. Seven centers tested the standardized methods by FACS-isolating a specific crypt-based epithelial population (EpCAM(+)/CD44(+)) from murine small intestine. Genetic biomarkers for stem/progenitor (Lgr5 and Atoh 1) and differentiated cell lineages (lysozyme, mucin2, chromogranin A, and sucrase isomaltase) were interrogated in target and control populations to assess intra- and intercenter variability. Wilcoxon's rank sum test on gene expression levels showed limited intracenter variability between biological replicates. Principal component analysis demonstrated significant intercenter reproducibility among four centers. Analysis of data collected by standardized cell isolation methods and data reporting requirements readily identified methodological problems, indicating that standard reporting parameters facilitate post hoc error identification. These results indicate that the complexity of FACS isolation of target intestinal epithelial populations can be highly reproducible between biological replicates and different institutions by adherence to common cell isolation methods and FACS gating strategies. This study can be considered a foundation for continued method development and a starting point for investigators that are developing cell isolation expertise to study physiology and pathophysiology of the intestinal epithelium.
View details for DOI 10.1152/ajpgi.00481.2012
View details for Web of Science ID 000325809200002
View details for PubMedID 23928185
The intestinal stem cell markers Bmi1 and Lgr5 identify two functionally distinct populations
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2012; 109 (2): 466-471
A remarkable feature of regenerative processes is their ability to halt proliferation once an organ's structure has been restored. The Wnt signalling pathway is the major driving force for homeostatic self-renewal and regeneration in the mammalian intestine. However, the mechanisms that counterbalance Wnt-driven proliferation are poorly understood. Here we demonstrate in mice and humans that yes-associated protein 1 (YAP; also known as YAP1)--a protein known for its powerful growth-inducing and oncogenic properties--has an unexpected growth-suppressive function, restricting Wnt signals during intestinal regeneration. Transgenic expression of YAP reduces Wnt target gene expression and results in the rapid loss of intestinal crypts. In addition, loss of YAP results in Wnt hypersensitivity during regeneration, leading to hyperplasia, expansion of intestinal stem cells and niche cells, and formation of ectopic crypts and microadenomas. We find that cytoplasmic YAP restricts elevated Wnt signalling independently of the AXIN-APC-GSK-3? complex partly by limiting the activity of dishevelled (DVL). DVL signals in the nucleus of intestinal stem cells, and its forced expression leads to enhanced Wnt signalling in crypts. YAP dampens Wnt signals by restricting DVL nuclear translocation during regenerative growth. Finally, we provide evidence that YAP is silenced in a subset of highly aggressive and undifferentiated human colorectal carcinomas, and that its expression can restrict the growth of colorectal carcinoma xenografts. Collectively, our work describes a novel mechanistic paradigm for how proliferative signals are counterbalanced in regenerating tissues. Additionally, our findings have important implications for the targeting of YAP in human malignancies.
View details for DOI 10.1038/nature11693
View details for Web of Science ID 000312933800040
View details for PubMedID 23178811
Structural insights into piRNA recognition by the human PIWI-like 1 PAZ domain
PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS
2011; 79 (6): 2004-2009
Use of the Colonoscope Training Model with the Colonoscope 3D Imaging Probe Improved Trainee Colonoscopy Performance: A Pilot Study
DIGESTIVE DISEASES AND SCIENCES
2011; 56 (5): 1496-1502
The small intestine epithelium undergoes rapid and continuous regeneration supported by crypt intestinal stem cells (ISCs). Bmi1 and Lgr5 have been independently identified to mark long-lived multipotent ISCs by lineage tracing in mice; however, the functional distinctions between these two populations remain undefined. Here, we demonstrate that Bmi1 and Lgr5 mark two functionally distinct ISCs in vivo. Lgr5 marks mitotically active ISCs that exhibit exquisite sensitivity to canonical Wnt modulation, contribute robustly to homeostatic regeneration, and are quantitatively ablated by irradiation. In contrast, Bmi1 marks quiescent ISCs that are insensitive to Wnt perturbations, contribute weakly to homeostatic regeneration, and are resistant to high-dose radiation injury. After irradiation, however, the normally quiescent Bmi1(+) ISCs dramatically proliferate to clonally repopulate multiple contiguous crypts and villi. Clonogenic culture of isolated single Bmi1(+) ISCs yields long-lived self-renewing spheroids of intestinal epithelium that produce Lgr5-expressing cells, thereby establishing a lineage relationship between these two populations in vitro. Taken together, these data provide direct evidence that Bmi1 marks quiescent, injury-inducible reserve ISCs that exhibit striking functional distinctions from Lgr5(+) ISCs and support a model whereby distinct ISC populations facilitate homeostatic vs. injury-induced regeneration.
View details for DOI 10.1073/pnas.1118857109
View details for Web of Science ID 000298950200030
View details for PubMedID 22190486
Acting in good taste: nutrient sensors in the gut
2009; 58 (7): 897-898
TAGging the target for damage control
NATURE STRUCTURAL BIOLOGY
2002; 9 (9): 638-640
FRS2 PTB domain conformation regulates interactions with divergent neurotrophic receptors
JOURNAL OF BIOLOGICAL CHEMISTRY
2002; 277 (19): 17088-17094
Colonoscopy insertion is difficult to teach due to the inability of current training models to provide realistic tactile sensation with simultaneous three-dimensional (3D) colonoscope display.To assess the influence of a simulator consisting of a colon model coupled with 3D instrument visualization on trainee colonoscopy performance.Pilot study using the simulator model with three trainees who were not proficient in colonoscopy. At random times over a 6-week period, trainees participated in an individualized half-day session using the Colonoscope Training Model and a colonoscope equipped with a 3D magnetic probe imaging system (ScopeGuide) in six standardized cases. A blinded supervising instructor graded patient-based colonoscopy performance over the 6-week period, and we independently analyzed the 2-week period before and after the intervention. We also measured cecal intubation and withdrawal times and medication requirements.Trainees performed 86 patient-based colonoscopies. Following the intervention, the colonoscopy performance score improved from 4.4 ± 2.3 to 5.9 ± 2.4 (p = 0.005). Trainees had a 76% cecal intubation rate following the session as compared to 43% before training (p = 0.004), while utilizing less time, 14 ± 7 versus 18 ± 11 min (p = 0.056) and less medication (p > 0.05).Colonoscopy simulation using the Colonoscope Training Model and the ScopeGuide produced an immediate and large effect on trainee colonoscopy performance.
View details for DOI 10.1007/s10620-011-1614-1
View details for Web of Science ID 000289899200033
View details for PubMedID 21409379
PTB or not PTB - that is the question
2002; 513 (1): 67-70
Membrane-anchored adaptor proteins FRS2alpha/beta (also known as SNT-1/2) mediate signaling of fibroblast growth factor receptors (FGFRs) and neurotrophin receptors (TRKs) through their N-terminal phosphotyrosine binding (PTB) domains. The FRS2 PTB domain recognizes tyrosine-phosphorylated TRKs at an NPXpY (where pY is phosphotyrosine) motif, whereas its constitutive association with FGFR involves a receptor juxtamembrane region lacking Tyr and Asn residues. Here we show by isothermal titration calorimetry that the FRS2alpha PTB domain binding to peptides derived from TRKs or FGFR is thermodynamically different. TRK binding is largely enthalpy-driven, whereas the FGFR interaction is governed by a favorable entropic contribution to the free energy of binding. Furthermore, our NMR spectral analysis suggests that disruption of an unstructured region C-terminal to the PTB domain alters local conformation and dynamics of the residues at the ligand-binding site, and that structural disruption of the beta8-strand directly weakens the PTB domain association with the FGFR ligand. Together, our new findings support a molecular mechanism by which conformational dynamics of the FRS2alpha PTB domain dictates its association with either fibroblast growth factor or neurotrophin receptors in neuronal development.
View details for DOI 10.1074/jbc.M107963200
View details for Web of Science ID 000175564500099
View details for PubMedID 11877385
Letter to the Editor: H-1, C-13 and N-15 resonance assignments of the SNT PTB domain in complex with FGFR1 peptide
JOURNAL OF BIOMOLECULAR NMR
2000; 18 (4): 371-372
Structural basis of SNT PTB domain interactions with distinct neurotrophic receptors
2000; 6 (4): 921-929
Phosphotyrosine binding (PTB) domains are structurally conserved modules found in proteins involved in numerous biological processes including signaling through cell-surface receptors and protein trafficking. While their original discovery is attributed to the recognition of phosphotyrosine in the context of NPXpY sequences -- a function distinct from that of the classical src homology 2 (SH2) domain -- recent studies show that these protein modules have much broader ligand binding specificities. These studies highlight the functional diversity of the PTB domain family as generalized protein interaction domains, and reinforce the concept that evolutionary changes of structural elements around the ligand binding site on a conserved structural core may endow these protein modules with the structural plasticity necessary for functional versatility.
View details for Web of Science ID 000174208800011
View details for PubMedID 11911882
SNT adaptor proteins transduce activation of fibroblast growth factor receptors (FGFRs) and neurotrophin receptors (TRKs) to common signaling targets. The SNT-1 phosphotyrosine binding (PTB) domain recognizes activated TRKs at a canonical NPXpY motif and, atypically, binds to nonphosphorylated FGFRs in a region lacking tyrosine or asparagine. Here, using NMR and mutational analyses, we show that the PTB domain utilizes distinct sets of amino acid residues to interact with FGFRs or TRKs in a mutually exclusive manner. The FGFR1 peptide wraps around the beta sandwich structure of the PTB domain, and its binding is possibly regulated by conformational change of a unique C-terminal beta strand in the protein. Our results suggest mechanisms by which SNTs serve as molecular switches to mediate the essential interplay between FGFR and TRK signaling during neuronal differentiation.
View details for Web of Science ID 000090136700015
View details for PubMedID 11090629