Biology subjects

Kieffer, T. J.

Publications and source records attributed to Kieffer, T. J..

4 recordsLinked to original sources

bioRxiv2024

Iterative sacrificial 3D printing and polymer casting to create complex vascular grafts and multi-compartment bioartificial organs

Several emerging strategies to engineer artificial organs employ 3D printing to create vascular templates to provide nutrients and oxygen to immobilized cells. Significant challenges emerge when considering clinical implementation such as immune rejection of allogeneic cell sources, as well as achieving adequate perfusion and integration with endogenous vasculature. We propose a method by which cell-laden hydrogels are molded around ready-made polymeric vascular templates created via 3D printing to create human-scale artificial organs with internal vasculature. We applied this technique to create bioartificial pancreas systems with up to 9 internal flow channels via sacrificial carbohydrate glass 3D printing, porogen-loaded polycarbonate polyurethane dip-coating, followed by casting cell-laden hydrogels around the vascular templates. We optimized porogen size and concentration to maximise the porosity of our scaffolds without compromising mechanical properties, resulting in suture retention strength and compliance respectively matching commercial vascular grafts and native vessels. Bioreactor perfusion studies showed survival and maturation of stem cell derived pancreatic islets without significant differences to traditional suspension culture protocols. Insulin response dynamics were rapid in response to a glucose challenge at the perfusion inlet. Transplantation of the devices as iliac arteriovenous shunts in nondiabetic pigs confirmed safety and patency. These results show promise for the development of an implantable vascularized pancreas for the treatment of type 1 diabetes and demonstrate how bioartificial organs with engineered vascular geometries can be designed for translational applications.

bioengineering↗

bioRxiv2023

Human A2-CAR T cells reject HLA-A2+ human islets transplanted into mice without inducing graft versus host disease

BackgroundType 1 diabetes (T1D) is an autoimmune disease characterised by T cell mediated destruction of pancreatic beta-cells. Islet transplantation is an effective therapy, but its success is limited by islet quality and availability along with the need for immunosuppression. New approaches include use of stem cell-derived insulin-producing cells and immunomodulatory therapies, but a limitation is the paucity of reproducible animal models in which interactions between human immune cells and insulin-producing cells can be studied without the complication of xenogeneic graft-versus-host disease (xGVHD). MethodsWe expressed an HLA-A2-specific chimeric antigen receptor (A2-CAR) in human CD4+ and CD8+ T cells and tested their ability to reject HLA-A2+ islets transplanted under the kidney capsule or anterior chamber of the eye of immunodeficient mice. T cell engraftment, islet function and xGVHD were assessed longitudinally. ResultsThe speed and consistency of A2-CAR T cells-mediated islet rejection varied depending on the number of A2-CAR T cells and the absence/presence of co-injected peripheral blood mononuclear cells (PBMCs). When <3 million A2-CAR T cells were injected, co-injection of PBMCs accelerated islet rejection but also induced xGVHD. In the absence of PBMCs, injection of 3 million A2-CAR T cells caused synchronous rejection of A2+ human islets within 1 week and without xGVHD for 12 weeks. ConclusionsInjection of A2-CAR T cells can be used to study rejection of human insulin-producing cells without the complication of xGVHD. The rapidity and synchrony of rejection will facilitate in vivo screening of new therapies designed to improve the success of isletreplacement therapies.

immunology↗

bioRxiv2019

Pancreas-specific miR-216a regulates proliferation and endocrine and exocrine cell function in vivo

Pancreas is a vital organ composed of exocrine and endocrine cells that aid digestion of food and regulate blood glucose levels. Perturbations in the function of pancreatic cells leads to the development of life-burdening and/or threatening diseases such as diabetes and pancreatic cancer. Thus, it is critical to understand the molecular check-points that maintain normal pancreas physiology. MicroRNAs (miRNAs) are small non-coding RNAs involved in regulating gene expression in normal and diseased tissues. Several miRNAs have tissue-specific patterns consistent with crucial functions in many biological processes. Yet, there is limited knowledge about the role of pancreas-specific miRNAs in pancreatic pathologies. Here, we report that miR-216a is a conserved, pancreas-specific miRNA that is expressed in both endocrine and exocrine cells. Deletion of miR-216a in mice leads to reduced {beta}-cell mass and a reduction in islet size under both chow and high-fat diet feeding conditions. We show that inhibition of miR-216a increases apoptosis and decreases cell proliferation in {beta}- and exocrine cells. Smad7 is upregulated in miR-216a deficient islets and cell cycle and proliferation are among the most significantly regulated biological processes in miR-216 knockout pancreata. Re-introduction of miR-216a in the pancreatic cancer line, PANC-1, increases cell migration more than 2-fold. In vivo, deletion of miR-216a in the pancreatic cancer prone mouse line KrasG12D;Ptf1aCreER inhibits the propensity of pancreatic cancer precursor lesions. Our study identifies miR-216a as an important pancreas-specific miRNA which may have implications for both diabetes and pancreatic cancer.

physiology↗

bioRxiv2019

Ins2 gene bursting activity defines a mature beta-cell state

Transcriptional and functional cellular specialization has been described for insulin-secreting {beta}-cells of the endocrine pancreas. However, it is not clear whether {beta}-cell heterogeneity is stable or reflects dynamic cellular states. We investigated the temporal kinetics of endogenous insulin gene activity using live cell imaging, with complementary experiments employing FACS and single cell RNA sequencing, in {beta}-cells from Ins2GFP knock-in mice. In vivo staining and FACS analysis of islets from Ins2GFP mice confirmed that at a given moment, ~25% of {beta}-cells exhibited significantly higher activity at the conserved insulin gene Ins2. Live cell imaging captured Ins2 gene activity dynamics in single {beta}-cells over days. Autocorrelation analysis revealed a subset of cells with oscillating behavior, with mean oscillation periods of 17 hours. Increased glucose concentrations stimulated more cells to oscillate and resulted in higher average Ins2 gene activity per cell. Single cell RNA sequencing showed that Ins2(GFP)HIGH {beta}-cells were enriched for markers of {beta}-cell maturity. Ins2(GFP)HIGH {beta}-cells were also significantly less viable at all glucose concentrations and in the context of ER stress. Collectively, our results demonstrate that the heterogeneity of insulin production, observed in mouse and human {beta}-cells, can be accounted for by dynamic states of insulin gene activity. BlurbPreviously reported pancreatic {beta}-cell heterogeneity reflects {beta}-cell state transitions.

cell biology↗