| 1. |
- Alshaikh, Ahmed Baker, et al.
(författare)
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Decellularization and recellularization of the ovary for bioengineering applications; studies in the mouse.
- 2020
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Ingår i: Reproductive biology and endocrinology : RB&E. - : Springer Science and Business Media LLC. - 1477-7827. ; 18:1
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Tidskriftsartikel (refereegranskat)abstract
- Fertility preservation is particularly challenging in young women diagnosed with hematopoietic cancers, as transplantation of cryopreserved ovarian cortex in these women carries the risk for re-introducing cancer cells. Therefore, the construction of a bioengineered ovary that can accommodate isolated small follicles was proposed as an alternative to minimize the risk of malignancy transmission. Various options for viable bioengineered scaffolds have been reported in the literature. Previously, we reported three protocols for producing mouse ovarian scaffolds with the decellularization technique. The present study examined these scaffolds further, specifically with regards to their extracellular composition, biocompatibility and ability to support recellularization with mesenchymal stem cells.Three decellularization protocols based on 0.5% sodium dodecyl sulfate (Protocol 1; P1), or 2% sodium deoxycholate (P2), or a combination of the two detergents (P3) were applied to produce three types of scaffolds. The levels of collagen, elastin and sulfated glycosaminoglycans (sGAGs) were quantified in the remaining extracellular matrix. Detailed immunofluorescence and scanning electron microscopy imaging were conducted to assess the morphology and recellularization efficiency of the constructs after 14days in vitro utilizing red fluorescent protein-labelled mesenchymal stem cells.All protocols efficiently removed the DNA while the elastin content was not significantly reduced during the procedures. The SDS-protocol (P1) reduced the sGAG and the collagen content more than the SDC-protocol (P2). All scaffolds were biocompatible and recellularization was successful, particularly in several P2-derived scaffolds. The cells were extensively distributed throughout the constructs, with a denser distribution observed towards the ovarian cortex. The cell density was not significantly different (400 to 550 cells/mm2) between scaffold types. However, there was a tendency towards a higher cell density in the SDC-derived constructs. Scanning electron microscope images showed fibrous scaffolds with a dense repopulated surface structure.While there were differences in the key structural macromolecules between protocols, all scaffolds were biocompatible and showed effective recellularization. The results indicate that our SDC-protocol might be better than our SDS-protocol. However, additional studies are necessary to determine their suitability for attachment of small follicles and folliculogenesis.
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| 2. |
- Kim, Jong Hyuk, et al.
(författare)
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Genomically Complex Human Angiosarcoma and Canine Hemangiosarcoma Establish Convergent Angiogenic Transcriptional Programs Driven by Novel Gene Fusions
- 2021
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Ingår i: Molecular Cancer Research. - : American Association For Cancer Research (AACR). - 1541-7786 .- 1557-3125. ; 19:5, s. 847-861
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Tidskriftsartikel (refereegranskat)abstract
- Sporadic angiosarcomas are aggressive vascular sarcomas whose rarity and genomic complexity present significant obstacles in deciphering the pathogenic significance of individual genetic alterations. Numerous fusion genes have been identified across multiple types of cancers, but their existence and significance remain unclear in sporadic angiosarcomas. In this study, we leveraged RNA-sequencing data from 13 human angiosarcomas and 76 spontaneous canine hemangiosarcomas to identify fusion genes associated with spontaneous vascular malignancies. Ten novel protein-coding fusion genes, including TEX2-PECAM1 and ATP8A2-FLT1, were identified in seven of the 13 human tumors, with two tumors showing mutations of TP53. HRAS and NRAS mutations were found in angiosarcomas without fusions or TP53 mutations. We found 15 novel protein-coding fusion genes including MYO16-PTK2, GABRA3-FLT1, and AKT3-XPNPEP1 in 11 of the 76 canine hemangiosarcomas; these fusion genes were seen exclusively in tumors of the angiogenic molecular subtype that contained recurrent mutations in TP53, PIK3CA, PIK3R1, and NRAS. In particular, fusion genes and mutations of TP53 cooccurred in tumors with higher frequency than expected by random chance, and they enriched gene signatures predicting activation of angiogenic pathways. Comparative transcriptomic analysis of human angiosarcomas and canine hemangiosarcomas identified shared molecular signatures associated with activation of PI3K/AKT/mTOR pathways. Our data suggest that genome instability induced by TP53 mutations might create a predisposition for fusion events that may contribute to tumor progression by promoting selection and/or enhancing fitness through activation of convergent angiogenic pathways in this vascular malignancy. Implications: This study shows that, while drive events of malignant vasoformative tumors of humans and dogs include diverse mutations and stochastic rearrangements that create novel fusion genes, convergent transcriptional programs govern the highly conserved morphologic organization and biological behavior of these tumors in both species.
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| 3. |
- Kim, Shin, et al.
(författare)
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Development of boron doped diamond electrodes material for heavy metal ion sensor with high sensitivity and durability
- 2023
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Ingår i: Journal of Materials Research and Technology. - : Elsevier BV. - 2238-7854 .- 2214-0697. ; 23, s. 1375-1385
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Tidskriftsartikel (refereegranskat)abstract
- We report on the optimized substrate pretreatment and deposition process conditions for boron-doped diamond (BDD) electrodes fabricated by hot-filament chemical vapor deposition (HFCVD). The optimized BDD electrode with a doping concentration of 8000 ppm showed high accuracy and precision in detecting Cd(II), Pb(II), and Cu(II) ions. In addition, this demonstrates excellent selectivity against external metal ions under the optimized stripping voltammetry measurement conditions. The detection limits of the target ions of Cd(II), Pb(II), and Cu(II) were 0.55 (+/- 0.05), 0.43 (+/- 0.04), and 0.74 (+/- 0.06) mg/L (S/N = 3), respectively. In real samples spiked with 100 mg/L Cd(II), Pb(II), and Cu(II), both the accuracy and precision of the BDD electrode were within 5%; the interference with organic matter was also negligible. The excellent selectivity and long-term stability indicate that the BDD electrode developed in this study are potentially useful for online water environment monitoring systems.
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| 4. |
- Kim, Yonghyo, et al.
(författare)
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Identification and validation of VEGFR2 kinase as a target of voacangine by a systematic combination of DARTS and MSI
- 2020
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Ingår i: Biomolecules. - : MDPI AG. - 2218-273X. ; 10:4
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Tidskriftsartikel (refereegranskat)abstract
- Although natural products are an important source of drugs and drug leads, identification and validation of their target proteins have proven difficult. Here, we report the development of a systematic strategy for target identification and validation employing drug affinity responsive target stability (DARTS) and mass spectrometry imaging (MSI) without modifying or labeling natural compounds. Through a validation step using curcumin, which targets aminopeptidase N (APN), we successfully standardized the systematic strategy. Using label-free voacangine, an antiangiogenic alkaloid molecule as the model natural compound, DARTS analysis revealed vascular endothelial growth factor receptor 2 (VEGFR2) as a target protein. Voacangine inhibits VEGFR2 kinase activity and its downstream signaling by binding to the kinase domain of VEGFR2, as was revealed by docking simulation. Through cell culture assays, voacangine was found to inhibit the growth of glioblastoma cells expressing high levels of VEGFR2. Specific localization of voacangine to tumor compartments in a glioblastoma xenograft mouse was revealed by MSI analysis. The overlap of histological images with the MSI signals for voacangine was intense in the tumor regions and showed colocalization of voacangine and VEGFR2 in the tumor tissues by immunofluorescence analysis of VEGFR2. The strategy employing DARTS and MSI to identify and validate the targets of a natural compound as demonstrated for voacangine in this study is expected to streamline the general approach of drug discovery and validation using other biomolecules including natural products.
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| 5. |
- Lee, Amos C., et al.
(författare)
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OPENchip : an on-chip in situ molecular profiling platform for gene expression analysis and oncogenic mutation detection in single circulating tumour cells
- 2020
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Ingår i: Lab on a Chip. - : Royal Society of Chemistry (RSC). - 1473-0197 .- 1473-0189. ; 20:5, s. 912-922
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Tidskriftsartikel (refereegranskat)abstract
- Liquid biopsy holds promise towards practical implementation of personalized theranostics of cancer. In particular, circulating tumour cells (CTCs) can provide clinically actionable information that can be directly linked to prognosis or therapy decisions. In this study, gene expression patterns and genetic mutations in single CTCs are simultaneously analysed by strategically combining microfluidic technology and in situ molecular profiling technique. Towards this, the development and demonstration of the OPENchip (On-chip Post-processing ENabling chip) platform for single CTC analysis by epithelial CTC enrichment and subsequent in situ molecular profiling is reported. For in situ molecular profiling, padlock probes that identify specific desired targets to examine biomarkers of clinical relevance in cancer diagnostics were designed and used to create libraries of rolling circle amplification products. We characterize the OPENchip in terms of its capture efficiency and capture purity, and validate the probe design using different cell lines. By integrating the obtained results, molecular analyses of CTCs from metastatic breast cancer (HER2 (ERBB2) gene expression and PIK3CA mutations) and metastatic pancreatic cancer (KRAS gene mutations) patients were demonstrated without any off-chip processes. The results substantiate the potential implementation of early molecular detection of cancer through sequencing-free liquid biopsy.
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| 6. |
- Padma, Arvind M., et al.
(författare)
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Decellularization protocol-dependent damage-associated molecular patterns in rat uterus scaffolds differentially affect the immune response after transplantation.
- 2021
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Ingår i: Journal of tissue engineering and regenerative medicine. - : Hindawi Limited. - 1932-7005 .- 1932-6254. ; 15:7, s. 674-685
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Tidskriftsartikel (refereegranskat)abstract
- Scaffolds derived from decellularized tissue possess many advantages for bioengineering applications, including for novel infertility treatments. However, the decellularization process results in allogenic-independent damage-associated molecular patterns (DAMPs). This field is poorly studied, in particular for uterus bioengineering applications. An increased knowledge concerning the immune system activation after transplantation of decellularized tissue will enable safer construct development and thereby accelerate translation from research to clinic. We therefore transplanted rat uterus scaffolds produced by three different decellularization protocols based on Triton X-100 (P1 and P2) or sodium deoxycholate (P3) in a syngeneic animal model and assessed the immune response towards DAMPs exposed by the decellularization process. Biopsies were retrieved on day 5, 15, and 30 post transplantation and immunohistochemistry-stained CD45+ (leucocytes), CD4+ (T-cells), CD8a+ (cytotoxic T-cells), CD22+ (B-cells), NCR1+ (NK-cells), CD68+ (pan-macrophages), and CD163+ (M2 macrophages) cells within the grafts were quantified. The gene expression for interferon γ, interleukin (IL)-1β, IL-2, IL-6, and tumor necrosis factor (TNF) eotaxin-2, RANTES, MCP-1, MIP-1α, MIP-3α, IL-8 were also measured. Scaffolds from P1 induced a rapid cell infiltration after transplantation, presumably induced by DNA-based DAMPs. However, this response was only transient. Protocol 3 derived scaffolds induced an early pro-inflammatory cytokine response at the transcript level which remained high throughout the study. This response may be caused by the stronger decellularization detergent that could expose more extracellular matrix-related DAMPs. However, earlier proteomics analysis also identified significantly more abundant heat shook proteins-related DAMPs in this scaffold type. Protocol 2 caused the least immunogenic scaffolds and should thus be the future focus for in vivo uterus bioengineering applications.
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| 7. |
- Padma, Arvind M., et al.
(författare)
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Immune response after allogeneic transplantation of decellularized uterine scaffolds in the rat.
- 2021
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Ingår i: Biomedical Materials. - 1748-605X. ; 16:4
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Tidskriftsartikel (refereegranskat)abstract
- Data on how the immune system reacts to decellularized scaffolds after implantation is scarce and difficult to interpret due to many heterogeneous parameters such as tissue-type match, decellularization method and treatment application. The engraftment of these scaffolds must prove safe and that they remain inert to the recipient's immune system to enable successful translational approaches and potential future clinical evaluation. Herein, we investigated the immune response after the engraftment of three decellularized scaffold types that previously showed potential to repair a uterine injury in the rat. Protocol (P) 1 and P2 were based on Triton-X100 and generated scaffolds containing 820 ng mg-1and 33 ng mg-1donor DNA per scaffold weight, respectively. Scaffolds obtained with a sodium deoxycholate-based protocol (P3) contained 160 ng donor DNA per mg tissue. The total number of infiltrating cells, and the population of CD45+leukocytes, CD4+T-cells, CD8a+cytotoxic T-cells, CD22+B-cells, NCR1+NK-cells, CD68+and CD163+macrophages were quantified on days 5, 15 and 30 after a subcutaneous allogenic (Lewis to Sprague Dawley) transplantation. Gene expression for the pro-inflammatory cytokines INF-γ, IL-1β, IL-2, IL-6 and TNF were also examined. P1 scaffolds triggered an early immune response that may had been negative for tissue regeneration but it was stabilized after 30 d. Conversely, P3 initiated a delayed immune response that appeared negative for scaffold survival. P2 scaffolds were the least immunogenic and remained similar to autologous tissue implants. Hence, an effective decellularization protocol based on a mild detergent was advantageous from an immunological perspective and appears the most promising for futurein vivouterus bioengineering applications.
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| 8. |
- Padma, Arvind M., et al.
(författare)
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Towards a bioengineered uterus: bioactive sheep uterus scaffolds are effectively recellularized by enzymatic preconditioning
- 2021
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Ingår i: Npj Regenerative Medicine. - : Springer Science and Business Media LLC. - 2057-3995. ; 6:1
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Tidskriftsartikel (refereegranskat)abstract
- Uterine factor infertility was considered incurable until recently when we reported the first successful live birth after uterus transplantation. However, risky donor surgery and immunosuppressive therapy are factors that may be avoided with bioengineering. For example, transplanted recellularized constructs derived from decellularized tissue restored fertility in rodent models and mandate translational studies. In this study, we decellularized whole sheep uterus with three different protocols using 0.5% sodium dodecyl sulfate, 2% sodium deoxycholate (SDC) or 2% SDC, and 1% Triton X-100. Scaffolds were then assessed for bioactivity using the dorsal root ganglion and chorioallantoic membrane assays, and we found that all the uterus scaffolds exhibited growth factor activity that promoted neurogenesis and angiogenesis. Extensive recellularization optimization was conducted using multipotent sheep fetal stem cells and we report results from the following three in vitro conditions; (a) standard cell culturing conditions, (b) constructs cultured in transwells, and (c) scaffolds preconditioned with matrix metalloproteinase 2 and 9. The recellularization efficiency was improved short-term when transwells were used compared with standard culturing conditions. However, the recellularization efficiency in scaffolds preconditioned with matrix metalloproteinases was 200-300% better than the other strategies evaluated herein, independent of decellularization protocol. Hence, a major recellularization hurdle has been overcome with the improved recellularization strategies and in vitro platforms described herein. These results are an important milestone and should facilitate the production of large bioengineered grafts suitable for future in vivo applications in the sheep, which is an essential step before considering these principles in a clinical setting.
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| 9. |
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| 10. |
- Tiemann, Tom T, et al.
(författare)
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Towards uterus tissue engineering: a comparative study of sheep uterus decellularisation
- 2020
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Ingår i: Molecular human reproduction. - : Oxford University Press (OUP). - 1460-2407 .- 1360-9947. ; 26:3, s. 167-178
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Tidskriftsartikel (refereegranskat)abstract
- © The Author(s) 2020. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. Uterus tissue engineering may dismantle limitations in current uterus transplantation protocols. A uterine biomaterial populated with patient-derived cells could potentially serve as a graft to circumvent complicated surgery of live donors, immunosuppressive medication and rejection episodes. Repeated uterine bioengineering studies on rodents have shown promising results using decellularised scaffolds to restore fertility in a partially impaired uterus and now mandate experiments on larger and more human-like animal models. The aim of the presented studies was therefore to establish adequate protocols for scaffold generation and prepare for future in vivo sheep uterus bioengineering experiments. Three decellularisation protocols were developed using vascular perfusion through the uterine artery of whole sheep uteri obtained from slaughterhouse material. Decellularisation solutions used were based on 0.5% sodium dodecyl sulphate (Protocol 1) or 2% sodium deoxycholate (Protocol 2) or with a sequential perfusion of 2% sodium deoxycholate and 1% Triton X-100 (Protocol 3). The scaffolds were examined by histology, extracellular matrix quantification, evaluation of mechanical properties and the ability to support foetal sheep stem cells after recellularisation. We showed that a sheep uterus can successfully be decellularised while maintaining a high integrity of the extracellular components. Uteri perfused with sodium deoxycholate (Protocol 2) were the most favourable treatment in our study based on quantifications. However, all scaffolds supported stem cells for 2weeks in vitro and showed no cytotoxicity signs. Cells continued to express markers for proliferation and maintained their undifferentiated phenotype. Hence, this study reports three valuable decellularisation protocols for future in vivo sheep uterus bioengineering experiments.
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