16 research outputs found
Developments in stem cell-derived islet replacement therapy for treating type 1 diabetes
The generation of islet-like endocrine clusters from human pluripotent stem cells (hPSCs) has the potential to provide an unlimited source of insulin-producing β cells for the treatment of diabetes. In order for this cell therapy to become widely adopted, highly functional and well-characterized stem cell-derived islets (SC-islets) need to be manufactured at scale. Furthermore, successful SC-islet replacement strategies should prevent significant cell loss immediately following transplantation and avoid long-term immune rejection. This review highlights the most recent advances in the generation and characterization of highly functional SC-islets as well as strategies to ensure graft viability and safety after transplantation
Single-nucleus multi-omics of human stem cell-derived islets identifies deficiencies in lineage specification
Insulin-producing β cells created from human pluripotent stem cells have potential as a therapy for insulin-dependent diabetes, but human pluripotent stem cell-derived islets (SC-islets) still differ from their in vivo counterparts. To better understand the state of cell types within SC-islets and identify lineage specification deficiencies, we used single-nucleus multi-omic sequencing to analyse chromatin accessibility and transcriptional profiles of SC-islets and primary human islets. Here we provide an analysis that enabled the derivation of gene lists and activity for identifying each SC-islet cell type compared with primary islets. Within SC-islets, we found that the difference between β cells and awry enterochromaffin-like cells is a gradient of cell states rather than a stark difference in identity. Furthermore, transplantation of SC-islets in vivo improved cellular identities overtime, while long-term in vitro culture did not. Collectively, our results highlight the importance of chromatin and transcriptional landscapes during islet cell specification and maturation
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Engineering Microfluidic Systems to Recapitulate Human Physiology
The overarching goal of this dissertation is to engineer cell culture platforms that recapitulate dynamic in vivo microenvironments and enable functional readouts that mimic organ-level physiology. Specifically, efforts were focused on developing novel dynamic cell culture devices, known as organs-on-chips, and integrated platforms to facilitate their use. Despite the potential of organs-on-chips to address challenging problems in biomedical research, the high technical skill required to fabricate and operate these devices has hindered their widespread adoption. An iterative design, build, test methodology was applied to the research and development of microfluidic devices and automated platforms. The dynamics of glucose stimulated insulin secretion function of pancreatic islets informed the initial design of the microfluidic device for organoids. A microfluidic device to recreate biologic barrier functions was originally inspired by the pressure driven filtration that occurs within the kidney glomeruli. These devices were built through subtractive rapid prototyping of noncytotoxic plastic. Human cells were incorporated into the devices. Microfluidic pumps were utilized to generate dynamic flow. The organs-on-chips were then tested to validate cell viability under dynamic culture conditions and the ability to model organ-level functional readouts. Finally, an integrated platform was developed to automate dynamic culture and functional assessments. Together, this research demonstrates that dynamic physiological processes can be modeled in vitro through the development organ-on-chip technology.</p
Microelectrode Array based Functional Testing of Pancreatic Islet Cells
Electrophysiological techniques to characterize the functionality of islets of Langerhans have been limited to short-term, one-time recordings such as a patch clamp recording. We describe the use of microelectrode arrays (MEAs) to better understand the electrophysiology of dissociated islet cells in response to glucose in a real-time, non-invasive method over prolonged culture periods. Human islets were dissociated into singular cells and seeded onto MEA, which were cultured for up to 7 days. Immunofluorescent imaging revealed that several cellular subtypes of islets; β, δ, and γ cells were present after dissociation. At days 1, 3, 5, and 7 of culture, MEA recordings captured higher electrical activities of islet cells under 16.7 mM glucose (high glucose) than 1.1 mM glucose (low glucose) conditions. The fraction of the plateau phase (FOPP), which is the fraction of time with spiking activity recorded using the MEA, consistently showed distinguishably greater percentages of spiking activity with high glucose compared to the low glucose for all culture days. In parallel, glucose stimulated insulin secretion was measured revealing a diminished insulin response after day 3 of culture. Additionally, MEA spiking profiles were similar to the time course of insulin response when glucose concentration is switched from 1.1 to 16.7 mM. Our analyses suggest that extracellular recordings of dissociated islet cells using MEA is an effective approach to rapidly assess islet functionality, and could supplement standard assays such as glucose stimulate insulin response
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Microelectrode Array based Functional Testing of Pancreatic Islet Cells
Electrophysiological techniques to characterize the functionality of islets of Langerhans have been limited to short-term, one-time recordings such as a patch clamp recording. We describe the use of microelectrode arrays (MEAs) to better understand the electrophysiology of dissociated islet cells in response to glucose in a real-time, non-invasive method over prolonged culture periods. Human islets were dissociated into singular cells and seeded onto MEA, which were cultured for up to 7 days. Immunofluorescent imaging revealed that several cellular subtypes of islets; β, δ, and γ cells were present after dissociation. At days 1, 3, 5, and 7 of culture, MEA recordings captured higher electrical activities of islet cells under 16.7 mM glucose (high glucose) than 1.1 mM glucose (low glucose) conditions. The fraction of the plateau phase (FOPP), which is the fraction of time with spiking activity recorded using the MEA, consistently showed distinguishably greater percentages of spiking activity with high glucose compared to the low glucose for all culture days. In parallel, glucose stimulated insulin secretion was measured revealing a diminished insulin response after day 3 of culture. Additionally, MEA spiking profiles were similar to the time course of insulin response when glucose concentration is switched from 1.1 to 16.7 mM. Our analyses suggest that extracellular recordings of dissociated islet cells using MEA is an effective approach to rapidly assess islet functionality, and could supplement standard assays such as glucose stimulate insulin response
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Roadblocks confronting widespread dissemination and deployment of Organs on Chips
Organ on Chip platforms hold significant promise as alternatives to animal models or traditional cell cultures, both of which poorly recapitulate human pathophysiology and human level responses. Within the last 15 years, we have witnessed seminal scientific developments from academic laboratories, a flurry of startups and investments, and a genuine interest from pharmaceutical industry as well as regulatory authorities to translate these platforms. This Perspective identifies several fundamental design and process features that may act as roadblocks that prevent widespread dissemination and deployment of these systems, and provides a roadmap to help position this technology in mainstream drug discovery.
Organ on Chip platforms hold significant promise as alternatives to traditional animal models or cell cultures. In this Perspective, the authors examine the barriers that prevent widespread dissemination and deployment of these systems
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Comparison of temperature change and resulting ablation size induced by a 902-928 MHz and a 2450 MHz microwave ablation system in in-vivo porcine kidneys
Introduction: Microwave ablation (MWA) uses heat to ablate undesired tissue. Development of pre-planning algorithms for MWA of small renal masses requires understanding of microwave-tissue interactions at different operating parameters. The objective of this study was to compare the performance of two MWA systems in in-vivo porcine kidneys.
Methods: Five ablations were performed using a 902-928 MHz system (24 W, 5 min) and a 2450 MHz system (180 W, 2 min). Nonlinear regression analysis of temperature changes measured 5 mm from the antenna axis was completed for the initial 10 s of ablation using the power equation
and after the inflection point using an exponential equation. Thermal damage was calculated using the Arrhenius equation. Long and short axis ablation diameters were measured.
Results: The average 'a' varied significantly between systems (902-928 MHz: 0.0299 ± 0.027, 2450 MHz: 0.1598 ± 0.158), indicating proportionality to the heat source, but 'b' did not (902-928 MHz: 1.910 ± 0.372, 2450 MHz: 2.039 ± 0.366), signifying tissue type dependence. Past the inflection point, average steady-state temperature increases were similar between systems but reached more quickly with the 2450 MHz system. Complete damage was reached at 5 mm for both systems. The 2450 MHz system produced significantly larger short axis ablations (902-928 MHz: 2.40 ± 0.54 cm, 2450 MHz: 3.32 ± 0.41cm).
Conclusion: The 2450 MHz system achieved similar steady state temperature increases compared to the 902-928 MHz system, but more quickly due to higher output power. Further investigations using various treatment parameters and precise thermal sensor placement are warranted to refine equation parameters for the development of an ablation model
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Engineered Microenvironments for Maturation of Stem Cell Derived Cardiac Myocytes
Through the use of stem cell-derived cardiac myocytes, tissue-engineered human myocardial constructs are poised for modeling normal and diseased physiology of the heart, as well as discovery of novel drugs and therapeutic targets in a human relevant manner. This review highlights the recent bioengineering efforts to recapitulate microenvironmental cues to further the maturation state of newly differentiated cardiac myocytes. These techniques include long-term culture, co-culture, exposure to mechanical stimuli, 3D culture, cell-matrix interactions, and electrical stimulation. Each of these methods has produced various degrees of maturation; however, a standardized measure for cardiomyocyte maturation is not yet widely accepted by the scientific community
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Integrated platform for operating and interrogating organs-on-chips (Electronic supplementary information (ESI) available. See DOI: 10.1039/c9ay01663e)
The development of microphysiological systems, also known as organs-on-chips, has highlighted the need for improved in vitro models for drug discovery. However, the highly specialized skillset required to design, build, and operate these systems has hindered adoption. Here, we describe an integrated platform that enables both continuous perifusion culture and dynamic cell secretion assays. We also developed a graphical user interface that allows the user to not only control the hardware components, but also define automated programs. The system's functionality was validated by maintaining pancreatic islets in culture under continuous perifusion for 24 hours, then performing a functional glucose stimulated insulin secretion assay. We demonstrate that islets remained both viable and functional on the platform with minimal user involvement
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Evaluating Vascularization of Heterotopic Islet Constructs for Type 1 Diabetes Using an In Vitro Platform
Type 1 diabetes (T1D) results from the autoimmune destruction of β-cells within the pancreatic islets of Langerhans. Clinical islet transplantation from healthy donors is proposed to ameliorate symptoms, improve quality of life, and enhance the life span of afflicted T1D patients. However, post-transplant outcomes are dependent on the survival of the transplanted islets, which relies on the engraftment of the islets with the recipient's vasculature among other factors. Treatment strategies to improve engraftment include combining islets with supporting cells including endothelial cells (EC) and mesenchymal stem cells (MSC), dynamic cells capable of robust immunomodulatory and vasculogenic effects. In this study, we developed an in vitro model of transplantation to investigate the cellular mechanisms that enhance rapid vascularization of heterotopic islet constructs. Self-assembled vascular beds of fluorescently stained EC served as reproducible in vitro transplantation sites. Heterotopic islet constructs composed of islets, EC, and MSC were transferred to vascular beds for modeling transplantation. Time-lapsed imaging was performed for analysis of the vascular bed remodeling for parameters of neo-vascularization. Moreover, sampling of media following modeled transplantation showed secretory profiles that were correlated with imaging analyses as well as with islet function using glucose-stimulated insulin secretion. Together, evidence revealed that heterotopic constructs consisting of islets, EC, and MSC exhibited the most rapid recruitment and robust branching of cells from the vascular beds suggesting enhanced neo-vascularization compared to islets alone and control constructs. Together, this evidence supports a promising cell transplantation strategy for T1D and also demonstrates a valuable tool for rapidly investigating candidate cellular therapies for transplantation