Objective
This project has two key goals: 1) Platform development: We will develop a platform for large-scale mapping and controlling of functional cell states within SC-islets (human stem cell-derived islets) using implanted electrical sensors & stimulators and spatially resolved gene expression profiling. 2) Discovery: we will apply this platform across multiple donor-derived SC-islets and isogenic lymphoid tissue to discover how SC-islet cell metabolic maturation states shape their interactions with immune cells.
SC-islets hold promise for Type 1 Diabetes (T1D) research and cell therapy, but often exhibit immature function, reflecting gaps in development, composition, and line-to-line variability. A central goal of this research is to address these gaps by leveraging the team’s ability to build “cyborg” SC-islets with embedded micro-electronic sensors and stimulators, profile cell function and gene expression states in the same cells, and use electrical stimulation to improve the glucose responsiveness of insulin- and glucagon-secreting SC-islet cells. Specifically, we aim to combine these advances into a tool for large-scale function-to-gene mappings across SC-islets derived from healthy, pre-diabetic, and T1D donors, using electrical stimulation to regularize function across donor lines. Then, we will apply this multimodal platform to define how the maturation state of SC-islet cells influences interactions with donor-matched and mismatched immune cells through co-culture and co-implantation models.
The first objective is to develop a platform to generate function-to-gene mappings across hundreds of SC-islet cells simultaneously. To achieve this, we will increase the sensor number and density in cyborg SC-islets, and adopt transparent electrodes that render sensor-contacting cells optically accessible, enabling imaging-based single-cell RNA sequencing to now be automated. We will then integrate sensor-adjacent fluorescent barcodes to map sensor positions and validate the new platform’s capacity to jointly profile cell electrical activity and gene expression in the same cells.
The second objective is to standardize SC-islet maturity across donor-derived stem cell lines and define how maturity shapes their interactions with immune cell co-cultures. To achieve this, we will apply electrical stimulation to iPSC-islets generated from 20 non-diabetic, pre-T1D, and T1D donors to regularize their glucose-stimulated function. Then, we will co-culture them with donor-matched or mismatched pancreatic lymph node and spleen lymphocytes to determine how the maturation state of insulin- and glucagon-secreting cells impacts T-cell infiltration and killing. Finally, we will generate function-to-gene mappings from SC islet-immune co-cultures, and combine them with continuous metabolic data, to identify signatures tied to immune-evasive or vulnerable states.
The final objective is to establish the platform in living animals, to chart the immune reactivity of transplanted SC-islets. To achieve this, we implant SC-islets with syringe-injectable, tissue-like electronics into non-immune mice to track real-time graft activity, followed by multimodal profiling to define the genetic and microenvironment changes associated with maturation post-transplant. Then we will co-implant cyborg islets with donor-matched or mismatched pancreatic lymphoid tissue fragments, enriched for islet-targeting lymphocytes, into non-immune mice to track real-time cell activity and survival. Finally, we will test how the pre-transplant maturation state influences immune reactivity profiles, integrating them with genetic and electrical signatures to define the cell molecular and functional states that govern immune susceptibility.
Together, these studies will forge new routes to not just standardize but to accelerate and expand functional the maturation of SC-islets, revealing principles to achieve hypo-immune grafting, to ultimately achieve broadly applicable cell therapy for T1D.
Background Rationale
Type 1 Diabetes (T1D) afflicts ~10 million people globally. T1D patients suffer chronic blood sugar dysregulation, which limits their life expectancy. Daily insulin injections can help control blood sugar, but cannot fully recapitulate natural control mechanisms, and can be cost-prohibitive, onerous, and carry side dangerous side effects. Islet transplants have proven successful as an alternative therapy for T1D complicated by dangerously low blood sugar. The federal drug administration thus approved Donislecel, an off-the-shelf T1D islet therapy. However, transplantable donor islets are in short supply, insufficient to meet the demand for millions of T1D patients.
Islets sourced from human pluripotent stem cells (SC-islets) instead offer an unlimited source of cells for T1D islet replacement therapy. Scientists at the University of Pennsylvania leading this project have pioneered methods for generating SC-islets in a scalable manner that mimic the formation and glucose-stimulated insulin secretion (GSIS) of natural islets, and upon transplant reverse diabetes in preclinical T1D models. A clinical trial of SC-islet transplantation recently showed that in >85% of recipients, need for insulin injections fell to <20 units/day within 120 days. Donor islet transplants achieve this within 12 days, however. The 10-fold slower therapeutic benefit of SC-islet grafts follows the fact that SC-islet GSIS function is immature relative to native islets. SC-islet cell composition and organization are also highly variable, causing inconsistent function. Improving and regularizing the manufacture and maturity of SC-islets is thus urgently needed to realize their potential as broadly translatable therapeutics.
This research team has several platforms to standardize SC-islet manufacture & maturity and model immunity:
• They generate SC-islets using bioreactors, which offer precise control over the culture environment and repeated sampling, enabling consistent differentiation yields across distinct stem cell lines.
• Following SC-islet generation, they purify insulin- and glucagon-secreting cells based on surface markers and re-assemble them at set ratios, generating SC-islets of defined composition.
• Their SC-islets gain robust, dynamic GSIS after 3 weeks of extended culture, and recreating daily feeding cycles then triggers rhythmic GSIS responses with higher stimulation capacity, offering an unprecedented in vitro model of human islet maturation.
• They engineered bioelectronics-implanted or “cyborg” SC-islets, enabling chronic & noninvasive assessment of cell electrical activities.
• To reveal the gene programs underlying islet cell functional states, they apply 3D spatial gene expression profiling to bioelectronics-implanted SC-islets, thereby linking cell electrical activities to gene expression programs in the same cells.
• They use implanted electrical actuators to deliver fixed or AI-driven dynamic electrical stimulation that enhance glucose responsiveness in SC-islet cells, pioneering a new frontier in T1D cell therapy—closed-loop control of SC-islet function.
• Leveraging syringe-injectable tissue-like electronics, successful subcutaneous islet transplant, and pancreatic lymph node banks enriched for islet-reactive white blood cells, they can now track graft activity and immune reactivity in real time.
SC-Islets sourced from induced pluripotent stem cells (iPSC-islets) also offer attractive models to study the determinants of islet autoimmunity. T1D onset co-occurs with islet functional maturation, suggesting that maturation potentiates liability to inflammation and autoimmunity in T1D. Alas, maturing T1D islets to test this premise are scarcely available from humans, while iPSC-islets offer limitless cells. This team has access to donor-matched iPSC and pancreatic lymph node banks, including from healthy, pre-T1D (autoantibody-positive), and T1D donors. Uniting this unique resource with their tools for tunable SC-islet manufacture and function, function-to-gene mappings, and lymphoid tissue co-implants will allow them to regularize iPSC-islet cell maturation states across donor lines to reveal how they steer islet-immune cell interactions. The work will thus deliver a large-scale platform that can be extended to other tissue constructs to enhance their function and test the impact on immune reactivity.
Description of Project
Type 1 diabetes (T1D) afflicts ~10 million people globally, including 2 million Americans. T1D patients suffer from loss of insulin-producing pancreatic islet cells resulting in chronic glucose dysregulation, which limits their life expectancy. Daily insulin injections can help control glucose, but cannot fully recapitulate natural control mechanisms. Moreover, taking insulin for life can be cost-prohibitive, onerous, and carry side effects such as risk of severe hypoglycemia and, if mismanaged, diabetic ketoacidosis. Islet transplants, sourced from cadaveric donors, can free T1D patients of insulin injections, and are now approved as cell therapy for severe T1D. Alas, transplantable cadaveric islets are scarce, insufficient to treat the millions of T1D patients.
Human stem cell-derived islets (SC-islets) offer a limitless cell source to achieve broad cell therapy for T1D. Yet, achieving functional maturity at transplant time, or inside the body, remains a critical barrier. This project proposes to tackle this barrier with a first-in-class system to chart and control SC-islet functional cell states, and to apply this system across donor-matched SC-islets and lymphocytes to investigate the hypothesis that islet cell maturity shapes islet-immune cell interactions.
To achieve this goal, the team recently developed several platforms for standardizing SC-islet production & function and modeling immune reactivity: (I) Scalable differentiation of 3D SC-islets of defined cell composition and tunable functional maturity. (II) A multi-modal platform combining 3D spatially resolved gene expression profiling & electronics-implanted SC-islets to link cell activities to underlying gene expression programs in the same cells. (III) Electrical stimulation to improve SC-islet glucose responsiveness. (IV) Syringe-injectable electronics for long-term graft studies. (V) donor-matched iPSC and pancreatic lymph node banks. (VI) Successful islet transplant beneath the skin.
Now, the team is uniting these tools to 1) Chart & control SC-islet maturation states, 2) Standardize them across multiple lines, and 3) Determine how they impact islet-immune interactions.
The project has three main parts: First, the team will expand the multimodal platform to be able to map cell activity and gene expression across hundreds of cells simultaneously. Second, the team will incorporate SC-islets derived from non-diabetic, at-risk, and T1D donors, using electrical stimulation to equalize their function, and co-culture them with donor-matched or mismatched white blood cells to determine the impact of SC-islet functional maturity on immune recognition & killing and the gene regulatory programs underlying it. Third, the team will establish the multimodal platform beneath the skin in mice, to track real-time grafted cell activity & immunity and study how maturity shapes immune reactivity, and what are the key cell molecular determinants, in a living animal model.
Each part has proof-of-principle, supporting the potential for combining them to significantly advance SC-islet functional and immune modeling capabilities.
If successful, this work will establish means to standardize the function of SC-islets and principles to modulate their immune reactivity to improve functional outcomes of T1D cell replacement therapy.
Anticipated Outcome
This platform development & discovery project seeks to develop a system for large-scale sensing and controlling of SC-islet (human stem cell-derived islets) functional cell states using implanted electrical sensors & stimulators and spatially-resolved gene expression profiling, and to apply this system across multiple donor-derived SC-islets and donor-matched lymphoid tissue to discover how SC-islet cell functional states shape interactions with immune cells.
If successful, the team will achieve several key outcomes:
1) The creation of a scalable platform for automated generation of spatially resolved single-cell gene-to-function mappings and electrical stimulation. By incorporating transparent electrodes, we expect the entire process—from adding cells to devices and recording and/or stimulating electrical activities to tissue clearing and imaging—to occur on a single platform, enabling profiling of up to 12 SC-islet samples in parallel.
2) Spatially resolved gene expression profiling in 12-well plate format (12 devices/plate) will allow large-scale interrogation of how gene regulatory events give rise to SC-islet cell functional states, and how these interact with tissue organization and composition to generate tissue-level outputs during functional maturation.
3) The platform will enable the team to test if T1D autoimmune responses are observable at advanced SC-islet maturation states, and if they require an external stressor as trigger, revealing the strengths and weaknesses of modeling self- vs. auto immune cell targeting of islet cells in the lab and inside the body.
4) Implementing the multi-modal platform for electrical sensing and stimulation inside the body will pioneer the ability to sense the function, dysfunction, and death of transplanted cells in real time.
5) The proposed studies will generate an atlas of SC-islet cell molecular and activity states during maturation in the lab and inside the body, and under self- and non-self immune recognition, including immune-protective metabolic dormancy states and tolerogenic signals.
6) The immune co-culture and co-implantation models will allow the team to test if metabolic suppression is a viable strategy to reduce stress reactivity and thus protect SC-islet from transplant-associated inflammation and immune rejection.
7) By harnessing bioelectricity to build next-generation SC-islets, the work will unlock means to standardize the function across many lines and principles to modulate their immune liability to improve the functional outcomes of T1D cell replacement therapy, a key priority to achieve broadly translatability.
8) The team will deliver a sensing and manipulation platform that can be used with other tissue constructs to enhance functional cell states and test the resulting impact on immune reactivity in the lab and inside the body.
By developing scalable systems that address line-to-line variability and inconsistency in SC-islet cell composition and batch-to-batch functionality, the team expects to build a foundation for future clinical studies to test the capabilities of bioelectronics-integrated platform in T1D individuals. These future studies will test using electrical stimulation to maximize SC-islet functionality before transplant; leveraging implanted sensors and wireless signal transmission to assess transplant efficacy in real time and for long-term graft viability and activity monitoring; and integrating sensing and stimulation with machine learning into a feedback control system that achieves closed-loop modulation of SC-islet function.
Relevance to T1D
Type 1 diabetes (T1D) afflicts ~10 million people globally, including 2 million Americans. T1D patients suffer from loss of insulin-producing pancreatic islet cells resulting in chronic glucose dysregulation, which limits their life expectancy. Daily insulin injections can help control glucose, but cannot fully recapitulate natural control mechanisms. Moreover, taking insulin for life can be cost-prohibitive, onerous, and carry side effects such as risk of severe hypoglycemia and, if mismanaged, diabetic ketoacidosis. Scientists at the University of Pennsylvania have completed a clinical trial of donor islet transplantation, with 87% of recipients reaching freedom from dangerously low blood sugar events 1 year post-transplant. The FDA thus approved Donislecel, an of-the-shelf donor islet therapy for T1D. Alas, transplantable donor islets are in short supply, insufficient to meet the demand for millions of T1D patients.
Human stem cell-derived islets (SC-islets) offer a limitless cell source to achieve broad cell therapy for T1D. Scientists at the University of Pennsylvania leading this project have pioneered methods for generating SC-islets in a scalable manner that mimic the formation and glucose-stimulated insulin secretion (GSIS) of natural islets, and upon transplant reverse diabetes in preclinical T1D models. A clinical trial of SC-islet transplantation recently showed that in >85% of recipients, need for insulin injections fell to <20 units/day within 120 days. Donor islet transplants achieve this within 12 days, however. The 10-fold slower therapeutic benefit of SC-islet grafts follows the fact that SC-islet GSIS function is immature relative to native islets. SC-islet cell composition and organization are also highly variable, causing inconsistent function. Improving and regularizing the manufacture and maturity of SC-islets is thus urgently needed to realize their potential as broadly translatable therapeutics.
This project proposes to address this urgent need with a first-in-class system to chart and control SC-islet functional cell states, and to apply this system across donor-matched SC-islets and lymphocytes to investigate the hypothesis that islet cell maturity shapes islet-immune cell interactions.
Current approaches to chart islet function—including imaging-based methods, intracellular electrophysiology, or conventional extracellular electrode arrays—cannot continuously track the activities of insulin-secreting and glucagon-secreting cells across intact 3D islets, as they are either invasive or toxic to cells and thus incompatible with long-term stable readouts or assay activity one plane at a time. The proposed system offers several advantages: It is noninvasive and continuous, and it captures 3D tissue-wide activities by using stretchable mesh electronics that are uniformly distributed during organogenesis and attain cell-level resolution. Current strategies to control SC-islet function—including through gene manipulation, cell enrichment, or drugs—remain constrained by scalability, line-to-line variability, and inconsistency in islet cell composition and batch-to-batch differentiation & GSIS function. The proposed system offers several advantages: it attains scalable, consistent differentiations across distinct lines, and sets composition and GSIS as controlled variables. The system is ideal to dissect how SC-islet maturity states impact immune reactivity, as it profiles these states in real time relative to cell survival / immune targeting and their underlying molecular control.
If successful, the scientists expect to build a foundation for future clinical studies to test the capabilities of bioelectronics-integrated platform in T1D individuals. These future studies will test using electrical stimulation to maximize SC-islet functionality before transplant; leveraging implanted sensors and wireless signal transmission to assess transplant efficacy in real time and for long-term graft viability and activity monitoring; and integrating sensing and stimulation with machine learning into a feedback control system that achieves closed-loop modulation of SC-islet function. Together, these tools and applications hold great promise to transform cell therapy for T1D.