TDK

Although Hirschsprung’s disease is a well-defined pathological condition characterized by abnormal development of the enteric nervous system, currently the only available treatment is the surgical removal of the affected intestinal segment. Therefore, regenerative medicine and the possibility of stem cell transplantation have received special attention for the treatment of Hirschsprung’s disease and other congenital neurointestinal diseases. Over the past decade, the in vitro culturing of 3D cell cultures, organoids, and spheroids has rapidly spread, aiming to reduce the use of animal experiments, which provide more realistic modeling of certain diseases’ pathogenesis, and increase the reliability of drug experiments. Experimental efforts to cure Hirschsprung’s disease focus on the culturing of gut tissue derived neural cell aggregates (enteric neurospheres) and their transplantation into the aganglionic intestinal segment. The first objective of my diploma work was to generate enteric neurospheres from intestinal tissue samples derived from chicken embryos. To optimize the process, I investigated the effects of adding GDNF, Noggin, and BMP-4 molecules. In summary, I successfully isolated and cultured enteric neurospheres from embryonic chicken hindguts. The neurospheres contained enteric neurons and glial cells, and their occurrence could be increased with GDNF. The enteric cells derived from neurospheres successfully colonized the aganglionic quail hindgut following transplantation. The addition of BMP-4, an important growth factor in enteric nervous system development, hindered the formation of enteric neurospheres. Conversely, Noggin potentially facilitates post-transplantation colonization, and previous results justify the use of Noggin to optimize the culturing conditions of neurospheres. Further experiments are required to examine its effects.

It is widely accepted that embryonic development of the bursa of Fabricius is impaired under the influence of various androgens. Specifically, testosterone propionate treatment of early chicken embryo results in chemical bursectomy. Previous developmental studies raised the possibility that testosterone affect the mesenchymal compartment of the bursa of Fabricius and during bursectomy primary impairment occurs in the mesenchyme. The general aim of my MSc student research project was to understand the developmental changes of the mesenchymal microenvironment of the bursa of Fabricius during testosterone treatment. Monoclonal antibodies specific for lympho-myeloid cell lineages (anti-CD45, EIVE-12, CSF1R and anti-chB6) and extracellular matrix proteins (anti-laminin, -fibronectin, -collagen type I, and -tenascin-C) has been used to study the bursal folliculogenesis and differentiation of mesenchymal compartment. Testosterone propionate treatment disrupted the colonization of hematopoietic cells into the bursa epithelium. Here, we also identify glycoproteins, including fibronectin and tenascin-C, as important regulators of bursa colonization. In developing chicken bursa of Fabricius fibronectin is strongly expressed in the mesenchyme, while tenascin-C expression occurs later when the follicular cortex start to differentiate. Our in vitro migration studies demonstrate that fibronectin is permissive whereas tenascin-C is inhibitory to B cell migration. Increased tenascin-C immunoreactivity have been identified is the bursa mesenchyme followed by testosterone treatment and is absent in control bursa. These studies demonstrate several novel roles for extracellular matrix in developing bursa of Fabricius and proposes a new hypothesis for tenascin-C produced in the developing bursa mesenchyme as inhibitory for the migration of B-cells.

The thesis can be accessed here. 

The enteric nervous system (ENS) in one of the most complex parts of the peripheral nervous system in vertebrates. The complexity of the ENS is due to the vast number of neurons and glial cells. Hirschsprung disease is a well-known congenital malformation of the ENS (~1/5000 live births), characterized by the congenital absence of the myenteric and submucosal plexuses of the colon, leading to intestinal obstruction and abnormal colonic distension in newborns. The surgical treatment of HSCR is imperfect, with long-term postoperative gastrointestinal complications like constipation and enterocolitis, therefore novel therapies are needed. Neural crest-derived progenitor cells of the enteric nervous system (ENSDCs) are a potential source of neurons and glia that could be used in future transplant therapies to help restore normal gut function in patients with HSCR. Transplantation of these stem cells offers an innovative approach for treating HSCR and other enteric neuropathies. We and others have developed techniques to isolate ENS progenitor cells from embryonic and postnatal mouse. These cells can be maintained and amplified in culture as floating neurospheres, which are aggregates of ENS derived stem cells and their differentiating neuronal and glial cell progeny. In this diploma work, ENS derived ENSDC progenitors were transplanted in a mouse model of a gut wall to analyze the cellular integration of the neurosphere derived cells and compare the ECM expression of the donor cells and host environment. It was found that enteric neurospheres express agrin. Furthermore, I found that in vitro treatment with agrin inhibited ENSDC migration. After 1-week the grafted ENSDCs were visualized by Wnt1;tdT transgenic red fluorescence combined with agrin and CS-56 specific immunolabelling. After co-staining with agrin specific antibody, I found that Wnt1 mouse derived ENSDCs strongly produce agrin, while chondroitin sulphate specific CS-56 antigen was also upregulated around transplanted neurospheres. My study adds agrin and CS-56 to the list of the inhibitory ECM which ENSDCs interact with during cell transplantation experiments. My findings suggest that agrin production prevents cell emigration from the transplanted neurospheres. Similarly, CS-56 antigen is highly secreted from the gut wall after transplantation, which further strengthens the inhibitory environment. These observations should be leveraged to develop novel cell transplantation protocols where inhibitory ECM molecules are degraded from the stem cell environment.

The thesis can be accessed here. 

The bursa of Fabricius is the primary limfoid organ of birds, where B-cell proliferation and differentiation takes place in the follicles, which is the basic structural and functional units. The follicles are composed of two distinct compartments, the ectodermal-derived medulla and the mesodermal-derived cortex. Although the histology of the follicular medulla required for B-cell maturation has been characterised in detail, the tissue and molecular composition of the ontogenetically later emerging cortical region is not known. In my thesis work, cellular and molecular analysis of the follicular cortex was performed by immunocytochemical and histological methods, supplemented by cell culture and embryo manipulation techniques. In contrast to medullary B-cells expressing IgM+ molecules, cortical B-cells are CXCR4 positive but do not express IgM. No dendritic cells are present in the follicular cortex, although CSF1R+/TIM4+/Lep100+ macrophages are evenly distributed. The lympho-myeloid cell-rich cortex is underpinned by cortical reticulum cells expressing desmin and vimentin, which produce a network of collagen (type I, type III, IV, VI), glycoprotein (laminin, fibronectin, fibrillin, tenascin-C) and proteoglycan-rich extracellular matrix. Detailed characterization of the extracellular matrix revealed that while most of the matrix proteins are expressed in the early embryonic bursa, the expression of tenascin-C is first observed 4-5 days before hatching. After hatching, cortical-specific tenascin-C is concentrated around the capillaries. In vitro studies of the function of tenascin-C have demonstrated that it represents an inhibitory environment for B-cell migration. Similarly, in vivo RCAS-Shh retroviral vectorinduced overexpression of tenascin-C inhibits B-cell colonization of developing follicles. In summary, 1) development of the follicle cortex starts at 5 days before hatching; 2) the B-cell population of the cortical follicle shows a specific Bu-1+/CXCR4+/IgM- expression pattern, and the follicle lacks CSF1R+ dendritic cells and has a CSF1R+/TIM4+/Lep100+ macrophage population. 3) The scaffold of the cortical tissue is composed of mesenchymal reticulum cells that produce a tenascin-C rich extracellular matrix. 4) In vivo and in vitro experiments show that tenascin-C provides an inhibitory environment for B cell migration.

The avian bursa of Fabricius (BF) is a primary lymphoid organ, critical to normal B lymphocyte development. During embryogenesis the epithelial anlage of the BF emerges as a diverticulum of the cloaca surrounded by undifferentiated mesenchyme. While it is believed that CD45+ hematopoietic stem cells colonize the epithelial-mesenchymal primordium that would provide a selective microenvironment for B cell precursor expansion, it is more likely that separate B-cell, macrophage and dendritic cell precursors colonize the mesenchyme, and some precursors migrate to the surface epithelium and initiate lymphoid follicle bud formation. The goal of this project is to characterize the developmental mechanisms of lymphoid follicle formation using a large panel of monoclonal antibodies (mAbs) specific for leukocytes (CD45), B cells (chB6, EIVE12), macrophages (TIM4) and bursal dendritic cells (CSF1R). The staining of embryonic BF by these mAbs helps to distinguish between three different lineages of hematopoietic cells. CD45+/EIVE12+ cells were first observed in the BF rudiment and many of them enter the surface epithelium to induce follicle bud formation. This will be colonized by the second cell type that belong to the CSF1R+/TIM4+ population, followed by chB6+ B cell precursors. In conclusion, we could determine three different types of precursors which colonize the embryonic BF, indicating that there is a pre-bursal segregation between these blood-borne cell lineages. Using chick-duck chimeras, we demonstrate that the first cell types which enter the bursal epithelium are not the dendritic/macrophages or B cell precursors, but are a transient lymphoid bud inducer cell population whose primary role is to induce follicle bud formation.

During the gastrointestinal tract development neural crest derived cells migrate into the gut’s mesenchyme where they form the enteric nervous system (ENS) components: submucosal and myenteric plexuses. If the enteric neural crest (NC) migration is perturbed in the developing embryo, aganglionic colon and Hirschsprung’s disease (HD) is formed. According to the earlier enteric nervous system research, embryonic ceca derived factors play an important role in the development of colonic nervous system. Embryo manipulation studies showed that the BMP4 morphogenetic factor occurs firstly in the ceca, later along the gut and plays a role in the normal development of plexuses. In our work we developed an organ culture technique, where we can observe the effect of a BMP antagonist, Noggin in the developing enteric nervous system. The organ explants were 5 days old embryonic gut segments. According to our former immunohistochemical (IHC) results, the population of neural crest cells begin to colonize the ceca in this stage. The isolated gut segments were cultured 48 hours long. To the treatment the migration of neural crest cells stopped and the myenteric and submucosal ganglia occurred just at the proximal colon. We have also observed, as soon as the „wavefront” cells (stem cells of the migrating neural crest derived cells) reached the ceca their proliferation increased. When we microsurgically ablated the ceca and these guts were cultured as earlier, abnormal enteric nervous system developed. Our results show, that the disturbed BMP signaling in chicken embryonic gut cultures eventuate irregular enteric nervous system development, which phenotype is very similar to hypo- and aganglionotic colon and Hirschsprung’s disease.

Infectious bursal disease (IBD) is an immunosuppressive illness of young chickens caused by IBD virus. The infection targets the bursa of Fabricius, the primary lymphoid organ of the birds, where it causes the destruction of the medulla and the depletion of B-lymphocytes. Previous observations using IBDV specific antibodies strongly suggested that, at the very beginning of the infection the bursal secretory dendritic cells (BSDCs) in the bursal lymphoid follicle are the primary targets of IBDV, and B cells are secondarily infected. Despite the important immunological role of the BSDCs, their identification is still difficult, mostly due to the absence of lineage specific markers. The aim of my study was to characterize the staining pattern of a novel BSDC specific mouse monoclonal antibody (mAb) clone 10C12, on the IBDV infected bursa of Fabricius. The immunocytochemistry staining showed that 10C12 mAb specifically stains BSDCs during IBDV infection. 10C12 is a more suitable marker for tracking BSDCs in IBDV infected bursa of Fabricius than previously used vimentin, which in addition to BSDC strongly stained the connective tissue. Double immunofluorescent staining analyzed with confocal microscopy supports the idea that in early stages of IBDV infection the 10C12+ BSDCs are the primary target cells for the virus. Conclusion: 10C12 mAb is a suitable molecular marker for detecting BSDCs both in healthy and IBDV infected bursa of Fabricius. Furthermore, introducing novel BSDC specific antibodies will greatly enhance our understanding of BSDC in host immune response to viral infections.

The domesticated chicken is widely used animal model in vertebrate developmental biology. The bursa of Fabricius is a primary lymphoepithelial organ of birds, that is responsible for B lymphocyte maturation. This discovery in chicken resulted in the emergence of avian immunology in 1970s. Besides its scientific role chicken is the most important protein source in human nutrition. Avian immunology is almost equal to immunology of domesticated chickens, therefore monoclonal and polyclonal antibodies against avian lymphomyeloid antigens have been produced mainly against chicken. Characterization of immune cells in wild birds could lead to a better understanding of the immune system. Here, we have described a novel guinea fowl lymphocyte specific antigen, recognized by our monoclonal antibody, designated 7H3. During the immunohistochemical characterization of the 7H3 monoclonal antibody the supernatant recognized the immature and mature T cells in the adult thymus, while in the bursa of Fabricius only isolated group of cells of the follicular cortex were positive. The medullary B lymphocytes of the bursa of Fabricius were negative for 7H3. The T-dependent areas of the peripheral lymphoid organs also express the 7H3 antigen. Macrophage, dendritic cell and platelet markers did not stain the 7H3+ (positive) cells. During embryonic development the first 7H3+ cells appeared in the yolk sac blood islands of 2-day-old guinea fowl embryo, and later were detected in embryonic circulation. In order to confirm the hemopoietic specifity of 7H3, co-stainings with CD45 were carried out. However the two antibodies recognized the same cell type in the 5-day-old guinea fowl embryo, the expression of 7H3 antigen preceded the appearance of the classical hematopoietic cell specific CD45 molecule. The 7H3 immunoreactive product showed a fine granular pattern accumulating in the cell membrane. Immunoblotting with the 7H3 antibody recognized protein bands at 70 and 140 kDa in lysates of adult guinea fowl thymus. In conclusion: The first 7H3 + cells appear in the yolk sac blood islands and later the 7H3+ hematopoietic cell can also be detected in embryonic blood. In adult guinea fowl the 7H3 antibody recognizes T lymphocytes of the thymus and peripheral lymphoid organs and a subpopulation of cortical B lymphocytes in the bursal follicles. Based on these result, we assume that the 7H3 antibody labels a new differentiation antigen, and can be a useful marker to characterize the hematopoietic cells and B lymphocytes of the bursa of Fabricius.

The GALT of the chicken is made of solitary lymphoid cells and aggregates forming nodules or organs for example the cecal tonsil and bursa of fabricius. The cecal tonsil is the largest of these chicken gut associated lymphoid tissues. After hatching the cecal tonsil undergoes most of its growth as is noted by the rapid expansion of all cell populations via the proliferation of the resident cells as well as a massive migration of circulating lymphocytes to the region. The lymphoid tissue also undergoes organization into germinal centers (B lymphocyte and dendritic cell rich regions) and inter-follicular areas (T lymphocyte and macrophage rich regions). The cecal tonsil is further organized into units each made up of a central ramified crypt, germinal centers and their interspersed interfollicular areas. The post-embryonic growth occurs rapidly, reaching adult conditions by the 4th day after hatching, the number of germinal centers continues to increase and reach a peak at week 8 after hatching. In conclusion the results of my immunohistochemical characterization of the cecal tonsil suggests that the development of the cecal tonsil starts at the 11TH day of embryogenesis and is hallmarked by the infiltration of the region by a CD45 expressing cell population, two days later (13TH embryonic day) several of these cells express the MHCII antigen. The accumulation of these cells in the presumptive site of the cecal tonsil occurs without an antigen stimulus which suggests that the location and development of the cecal tonsil is genetically determined, the actual gene/genes responsible for this is yet to be characterized The cell population within the cecal tonsil gradually increase due to the migration and proliferation of cells. The appearance MHCII and 74.3 expressing dendritic cells under the epithelium supports the notion that the formation of the cecal tonsil is initiated by the accumulation of CD45+ve, MHC II+ve and 74.3+ve cells which go on to induce the formation of the lymphoepithelium.

The thesis can be accessed here.