| 1. |
- Dekki Shalaly, Nancy, et al.
(författare)
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Silk matrices promote formation of insulin-secreting islet-like clusters
- 2016
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Ingår i: Biomaterials. - : Elsevier BV. - 0142-9612 .- 1878-5905. ; 90, s. 50-61
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Tidskriftsartikel (refereegranskat)abstract
- Ex vivo expansion of endocrine cells constitutes an interesting alternative to be able to match the unmet need of transplantable pancreatic islets. However, endocrine cells become fragile once removed from their extracellular matrix (ECM) and typically become senescent and loose insulin expression during conventional 2D culture. Herein we develop a protocol where 3D silk matrices functionalized with ECM-derived motifs are used for generation of insulin-secreting islet-like clusters from mouse and human primary cells. The obtained clusters were shown to attain an islet-like spheroid shape and to maintain functional insulin release upon glucose stimulation in vitro. Furthermore, in vivo imaging of transplanted murine clusters showed engraftment with increasing vessel formation during time. There was no sign of cell death and the clusters maintained or increased in size throughout the period, thus suggesting a suitable cluster size for transplantation.
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| 2. |
- Dekki Wenna, Nancy
(författare)
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Serum proteins in type 1 diabetes
- 2007
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- We have previously shown that there are patients with type 1 diabetes T1D whose sera interfere with cytoplasmic free Ca2+-concentration ([Ca2+]i), resulting in â-cell apoptosis. In this study the frequency of sera, that induces this effect, was found to be around 35% in T1D patients, and 25% in first degree relatives (FDR) of T1D patients. The effect occurred in subjects of different gender, age and ethnicity, and was not related to the presence of autoantibodies. Hence, in a defined subgroup of patients with T1D and FDR a defect Ca2+-handling may aggravate the autoimmune response and thereby the development of â-cell destruction, and it might be possible to interfere early on with Ca2+-induced â-cell apoptosis. Purification and fractionation of T1D sera, that affected [Ca2+]i, revealed that the protein that gave a higher increase in [Ca2+]i upon depolarization, was apolipoprotein CIII (apoCIII). ApoCIII increases the activity of voltage-gated Ca2+-channels. A proof for apoCIII being the responsible serum component was that the addition of an antibody against apoCIII abolished, not only the effects of apoCIII on [Ca2+]i and cell death, but most importantly the effects of T1D sera itself on â-cell function and survival. During the procedure to purify the serum factor, we observed a band at 14 kDa on SDS/PAGE that was stronger in T1D than control sera. The protein in the band was identified by sequence analysis to be the monomeric form of transthyretin (TTR). TTR is a transport protein and exists mainly as a tetramer in sera from healthy individuals. In T1D sera the concentration of TTR was decreased, whereas the concentration of the monomer was increased. Physiological concentrations of the tetrameric form of TTR was shown to affect the â-cell stimulus-secretion coupling, promoting glucose-induced increases in [Ca2+]i and insulin release, which resulted from a direct effect on glucoseinduced electrical activity and voltage-gated Ca2+-channels. The tetrameric form of TTR also protected against apoCIII induced â-cell death. The monomer was without effect on glucose-stimulated insulin release and â-cell death. Therefore, conversion of TTR tetramer to monomer may be involved in the development of â-cell failure in T1D. TTR was shown to bind to glucose regulated proteins (Grp), 78, 94 and 170 in both the membrane and cytosolic fractions of islet cell homogenates, and was internalized into the â-cell via a clathrin-dependent pathway, indicating the involvement of receptormediated endocytosis. The Grp complex of Grp78, 94 and 170 may serve as a plasma membrane protein responsible for the binding and uptake of TTR into the â-cell. These data may suggest that the effects of TTR on â-cell function and survival are due to intracellular effects of the protein. In conclusion, there is a group of patients with T1D and FDR that have sera that affects [Ca2+]i and â-cell survival. The serum factor responsible for these effects was identified to be apoCIII, which is increased in T1D sera. The concentration of TTR tetramer is decreased, whereas that of the TTR monomer is increased in T1D sera. The tetrameric form of TTR was shown to have a positive role in the â-cell stimulussecretion coupling. These data support the possibility to develop a diagnostic test to identify individuals at risk to be subjected to Ca2+-induced â-cell damage and thereby aggravation of the autoimmune response in T1D.
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| 3. |
- Johansson, Ulrika, 1974-, et al.
(författare)
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Assembly of functionalized silk together with cells to obtain proliferative 3D cultures integrated in a network of ECM-like microfibers
- 2019
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Ingår i: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 9, s. 1-13
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Tidskriftsartikel (refereegranskat)abstract
- Tissues are built of cells integrated in an extracellular matrix (ECM) which provides a three-dimensional (3D) microfiber network with specific sites for cell anchorage. By genetic engineering, motifs from the ECM can be functionally fused to recombinant silk proteins. Such a silk protein, FN-silk, which harbours a motif from fibronectin, has the ability to self-assemble into networks of microfibers under physiological-like conditions. Herein we describe a method by which mammalian cells are added to the silk solution before assembly, and thereby get uniformly integrated between the formed microfibers. In the resulting 3D scaffold, the cells are highly proliferative and spread out more efficiently than when encapsulated in a hydrogel. Elongated cells containing filamentous actin and defined focal adhesion points confirm proper cell attachment to the FN-silk. The cells remain viable in culture for at least 90 days. The method is also scalable to macro-sized 3D cultures. Silk microfibers formed in a bundle with integrated cells are both strong and extendable, with mechanical properties similar to that of artery walls. The described method enables differentiation of stem cells in 3D as well as facile co-culture of several different cell types. We show that inclusion of endothelial cells leads to the formation of vessel-like structures throughout the tissue constructs. Hence, silk-assembly in presence of cells constitutes a viable option for 3D culture of cells integrated in a ECM-like network, with potential as base for engineering of functional tissue.
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| 4. |
- Johansson, Ulrika, 1974-, et al.
(författare)
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Integration of Primary Endocrine Cells and Supportive Cells Using Functionalized Silk Promotes the Formation of Prevascularized Islet-like Clusters
- 2020
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Ingår i: ACS Biomaterials Science & Engineering. - : American Chemical Society (ACS). - 2373-9878. ; 6:2, s. 1186-1195
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Tidskriftsartikel (refereegranskat)abstract
- Pancreatic islet transplantation has not yet succeeded as an overall treatment for type 1 diabetes because of limited access to donor islets, as well as low efficacy and poor reproducibility of the current procedure. Herein, a method to create islets-like composite clusters (coclusters) from dispersed endocrine cells and supportive cells is described, attempting to improve compatibility with the recipient and more efficiently make use of the donor-derived material. To mimic the extracellular matrix environment, recombinant,spider silk functionalized with cell binding motifs are used as 3D support for the coclusters. A cell binding motif derived from fibronectin (FN) was found superior in promoting cell adherence, while a plain RGD-motif incorporated in the repetitive part of the silk protein (2R) increased the mobility and cluster formation of endocrine cells. Self-assembly of a mixture of FN/2R silk is utilized to integrate endocrine cells together with endothelial and mesenchymal cells into islet-like coclusters. Both xenogenic and allogenic versions of these coclusters were found to be viable and were able to respond to dynamic glucose stimulation with insulin release. Moreover, the endothelial cells were found to be colocalized with the endocrine cells, showing that the silk combined with supportive cells may promote vascularization. This method to engineer combined islet-like coclusters allows donor-derived endocrine cells to be surrounded by supportive cells from the recipient, which have the potential to further promote engraftment in the host and considerably reduce risk of rejection.
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| 5. |
- Johansson, Ulrika, 1974-, et al.
(författare)
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Pancreatic Islet Survival and Engraftment Is Promoted by Culture on Functionalized Spider Silk Matrices
- 2015
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 19:10, s. 1-21
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Tidskriftsartikel (refereegranskat)abstract
- Transplantation of pancreatic islets is one approach for treatment of diabetes, however, hampered by the low availability of viable islets. Islet isolation leads to disruption of the environment surrounding the endocrine cells, which contributes to eventual cell death. The reestablishment of this environment is vital, why we herein investigated the possibility of using recombinant spider silk to support islets in vitro after isolation. The spider silk protein 4RepCT was formulated into three different formats; 2D-film, fiber mesh and 3D-foam, in order to provide a matrix that can give the islets physical support in vitro. Moreover, cell-binding motifs from laminin were incorporated into the silk protein in order to create matrices that mimic the natural cell environment. Pancreatic mouse islets were thoroughly analyzed for adherence, necrosis and function after in vitro maintenance on the silk matrices. To investigate their suitability for transplantation, we utilized an eye model which allows in vivo imaging of engraftment. Interestingly, islets that had been maintained on silk foam during in vitro culture showed improved revascularization. This coincided with the observation of preserved islet architecture with endothelial cells present after in vitro culture on silk foam. Selected matrices were further evaluated for long-term preservation of human islets. Matrices with the cell-binding motif RGD improved human islet maintenance (from 36% to 79%) with preserved islets architecture and function for over 3 months in vitro. The islets established cell-matrix contacts and formed vessel-like structures along the silk. Moreover, RGD matrices promoted formation of new, insulin-positive islet-like clusters that were connected to the original islets via endothelial cells. On silk matrices with islets from younger donors (<35 year), the amount of newly formed islet-like clusters found after 1 month in culture were almost double compared to the initial number of islets added.
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| 6. |
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| 7. |
- Shalaly, Nancy Dekki, et al.
(författare)
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Positive Modulation of the Glycine Receptor by Means of Glycine Receptor-Binding Aptamers
- 2015
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Ingår i: Journal of Biomolecular Screening. - : Sage Publications. - 1087-0571 .- 1552-454X. ; 20:9, s. 1112-1123
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Tidskriftsartikel (refereegranskat)abstract
- According to the gate control theory of pain, the glycine receptors (GlyRs) are putative targets for development of therapeutic analgesics. A possible approach for novel analgesics is to develop a positive modulator of the glycine-activated Cl- channels. Unfortunately, there has been limited success in developing drug-like small molecules to study the impact of agonists or positive modulators on GlyRs. Eight RNA aptamers with low nanomolar affinity to GlyR1 were generated, and their pharmacological properties analyzed. Cytochemistry using fluorescein-labeled aptamers demonstrated GlyR1-dependent binding to the plasma membrane but also intracellular binding. Using a fluorescent membrane potential assay, we could identify five aptamers to be positive modulators. The positive modulation of one of the aptamers was confirmed by patch-clamp electrophysiology on L(tk) cells expressing GlyR1 and/or GlyR1. This aptamer potentiated whole-cell Cl- currents in the presence of low concentrations of glycine. To our knowledge, this is the first demonstration ever of RNA aptamers acting as positive modulators for an ion channel. We believe that these aptamers are unique and valuable tools for further studies of GlyR biology and possibly also as tools for assay development in identifying small-molecule agonists and positive modulators.
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| 8. |
- Widhe, Mona, et al.
(författare)
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A fibronectin mimetic motif improves integrin mediated cell biding to recombinant spider silk matrices
- 2016
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Ingår i: Biomaterials. - : Elsevier. - 0142-9612 .- 1878-5905. ; 74, s. 256-266
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Tidskriftsartikel (refereegranskat)abstract
- The cell binding motif RGD is the most widely used peptide to improve cell binding properties of various biomaterials, including recombinant spider silk. In this paper we use genetic engineering to further enhance the cell supportive capacity of spider silk by presenting the RGD motif as a turn loop, similar to the one found in fibronectin (FN), but in the silk stabilized by cysteines, and therefore denoted FNCC. Human primary cells cultured on FNCC-silk showed increased attachment, spreading, stress fiber formation and focal adhesions, not only compared to RGD-silk, but also to silk fused with linear controls of the RGD containing motif from fibronectin. Cell binding to FNCC-silk was shown to involve the alpha 5 beta 1 integrin, and to support proliferation and migration of keratinocytes. The FNCC-silk protein allowed efficient assembly, and could even be transformed into free standing films, on which keratinocytes could readily form a monolayer culture. The results hold promise for future applications within tissue engineering.
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