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    MANIPULATING GROWTH AND DIFFERENTIATION OF EMBRYONIC INTESTINE IN ORGAN CULTURE

    Coletta, Riccardo

    [Thesis]. Manchester, UK: The University of Manchester; 2017.

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    Abstract

    Background. An ex vivo experimental strategy replicating in vivo intestinal development would provide an accessible setting to study normal and dysmorphic biology, and would be a test bed for tissue engineering. Previous studies implicated transforming growth factor β1 (TGFβ1) in postnatal gut maturation and regeneration following injury, but its potential role in intestinal development is poorly understood. I firstly hypothesised that embryonic small intestine is able to heal after physical injury. To test this idea, I aimed to create an organ culture model using explants of embryonic jejunum. I secondly hypothesised that TGFβ1 affects embryonic small intestine growth and differentiation. Accordingly, I aimed to use the same organ culture model to determine potential effects of exogenous TGFβ1.Methods. Segments of mouse embryonic jejunum were isolated by dissection and placed on semipermeable platforms. They were fed with defined, serum free, media, in some cases supplemented with TGFβ1. Growth, differentiation and healing of explants were characterized and quantified using a battery of techniques that included whole mount imaging, histology, immunostaining and RNA arrays. TGFβ1 was measured in amniotic fluid by enzyme-linked immunosorbent assay. Groups were compared by statistical tests.Results. After three days of culture, jejunal rudiments differentiated from simple tubes into a more complex structures containing smooth muscle surrounding newly formed villi. Pairs of rudiments, linked by a thread, fused and formed a continuous single lumen, as assessed by trajectories of fluorescent dextrans injected into their distal ends. Functional continuity was confirmed by spontaneous waves of peristalsis crossing the point of fusion. In vivo, TGFβ receptors I and II were detected in embryonic longitudinal smooth muscle cells and, in organ culture, exogenous TGFβ1 induced differentiation of longitudinal smooth muscle. Microarray profiling showed that TGFβ1 increased smooth muscle associated transcripts in a dose-dependent manner. TGFβ1 protein was detected in amniotic fluid at a time when the embryonic small intestine was physiologically herniated.Conclusion. Embryonic jejunal segments can fuse to form a single functional organ when aided by a mechanical manipulation. By analogy with the requirement for exogenous TGFβ1 for smooth muscle differentiation in culture, the TGFβ1 protein that I demonstrated to be present in the amniotic fluid may enhance intestinal development when it is physiologically herniated in early gestation. Future studies of embryonic intestinal cultures should add TGFβ1 in the defined media to produce a more faithful model of in vivo muscle differentiation. In future, this model could be used to test whether other growth factors enhance intestinal growth, and so pave the way to novel biological treatments for short bowel syndrome, a devastating disease with a high mortality.

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    Bibliographic metadata

    Type of resource:
    Content type:
    Form of thesis:
    Type of submission:
    Degree type:
    Doctor of Philosophy
    Degree programme:
    PhD Medicine 3yr (CMB)
    Publication date:
    Location:
    Manchester, UK
    Total pages:
    258
    Abstract:
    Background. An ex vivo experimental strategy replicating in vivo intestinal development would provide an accessible setting to study normal and dysmorphic biology, and would be a test bed for tissue engineering. Previous studies implicated transforming growth factor β1 (TGFβ1) in postnatal gut maturation and regeneration following injury, but its potential role in intestinal development is poorly understood. I firstly hypothesised that embryonic small intestine is able to heal after physical injury. To test this idea, I aimed to create an organ culture model using explants of embryonic jejunum. I secondly hypothesised that TGFβ1 affects embryonic small intestine growth and differentiation. Accordingly, I aimed to use the same organ culture model to determine potential effects of exogenous TGFβ1.Methods. Segments of mouse embryonic jejunum were isolated by dissection and placed on semipermeable platforms. They were fed with defined, serum free, media, in some cases supplemented with TGFβ1. Growth, differentiation and healing of explants were characterized and quantified using a battery of techniques that included whole mount imaging, histology, immunostaining and RNA arrays. TGFβ1 was measured in amniotic fluid by enzyme-linked immunosorbent assay. Groups were compared by statistical tests.Results. After three days of culture, jejunal rudiments differentiated from simple tubes into a more complex structures containing smooth muscle surrounding newly formed villi. Pairs of rudiments, linked by a thread, fused and formed a continuous single lumen, as assessed by trajectories of fluorescent dextrans injected into their distal ends. Functional continuity was confirmed by spontaneous waves of peristalsis crossing the point of fusion. In vivo, TGFβ receptors I and II were detected in embryonic longitudinal smooth muscle cells and, in organ culture, exogenous TGFβ1 induced differentiation of longitudinal smooth muscle. Microarray profiling showed that TGFβ1 increased smooth muscle associated transcripts in a dose-dependent manner. TGFβ1 protein was detected in amniotic fluid at a time when the embryonic small intestine was physiologically herniated.Conclusion. Embryonic jejunal segments can fuse to form a single functional organ when aided by a mechanical manipulation. By analogy with the requirement for exogenous TGFβ1 for smooth muscle differentiation in culture, the TGFβ1 protein that I demonstrated to be present in the amniotic fluid may enhance intestinal development when it is physiologically herniated in early gestation. Future studies of embryonic intestinal cultures should add TGFβ1 in the defined media to produce a more faithful model of in vivo muscle differentiation. In future, this model could be used to test whether other growth factors enhance intestinal growth, and so pave the way to novel biological treatments for short bowel syndrome, a devastating disease with a high mortality.
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    Non-digital content not deposited electronically:
    PRINTED COPY OF PUBLICATIONS RELATED ON THESIS
    Thesis main supervisor(s):
    Thesis co-supervisor(s):
    Language:
    en

    Institutional metadata

    University researcher(s):
    Academic department(s):

    Record metadata

    Manchester eScholar ID:
    uk-ac-man-scw:308007
    Created by:
    Coletta, Riccardo
    Created:
    13th March, 2017, 11:05:55
    Last modified by:
    Coletta, Riccardo
    Last modified:
    6th April, 2017, 08:05:37

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