Biology subjects

Oberholzer, J.

Publications and source records attributed to Oberholzer, J..

6 recordsLinked to original sources

bioRxiv2025

Localized immunomodulation with cytokine-producing cells to mitigate host immune rejection responses in rodents and a non-human primate

The efficacy of cell-based therapeutics is often compromised by host immune recognition of implanted cells and biomaterials, resulting in fibrotic encapsulation and loss of function. Here, we address this challenge with an immunomodulatory cell-based therapy, in which alginate-encapsulated retinal pigment epithelial cells continuously secrete cytokines to locally modulate the implant microenvironment. In a healthy rodent model, the localized production of interleukin-10 (IL-10) or IL-12 from encapsulated cytokine-producing cells prevented host immune rejection and fibrosis of alginate capsules. Mechanistically, treatment was associated with reduced expression of pro-fibrotic genes and immune shifts consistent with macrophage and T-cell regulation, supporting a cytokine-mediated mitigation of foreign body response. In a diabetic murine model (streptozotocin-induced C57BL/6J), co-implantation of human islets with IL-10-producing cells attenuated pericapsular fibrosis, preserved islet viability, and restored normoglycemia for up to 100 days (4.76 times longer than islets alone). Significantly, IL-10-producing cells were also effective in enabling the durability and function of encapsulated cells in a healthy non-human primate, showing translational feasibility. Collectively, these findings suggest that localized cytokine delivery can reduce fibrotic encapsulation and support durable graft function, offering a path to lessen reliance on systemic immunosuppression in islets transplantation and other implantable biomaterial therapies. TeaserEncapsulated IL-10-producing cells locally suppress fibrosis and extend graft function in rodent models and a non-human primate.

bioengineering↗

bioRxiv2023

Phase-changing citrate macromolecule combats oxidative pancreatic islet damage, enables islet engraftment and function in the omentum

Clinical outcomes for total-pancreatectomy followed by intraportal islet autotransplantation (TP-IAT) to treat chronic pancreatitis (CP) patients are suboptimal due to the inflammatory state of the patients pancreas, oxidative tissue damage during the isolation process, and the harsh engraftment conditions in the livers vasculature, which include ischemia-reperfusion injury, and instant blood-mediated inflammatory reactions. We describe the use of the thermoresponsive, antioxidant macromolecule poly(polyethylene glycol citrate-co-N-isopropylacrylamide) (PPCN) to protect islet redox status and function in vitro and in vivo and to create a viable extrahepatic islet engraftment site in the abdomen. PPCN in aqueous media transitions from a liquid to an elastic hydrogel when exposed to body temperature via temperature-induced macromolecular self-assembly. Islets entrapped in the PPCN hydrogel and exposed to oxidative stress remain functional and support long-term euglycemia, in contrast to islets entrapped in a biologic scaffold (BS). When applied to the omentum of non-human primates (NHPs), PPCN is well-tolerated, safe, and mostly resorbed without fibrosis at 3 months post-implantation. To obtain autologous islets, a partial pancreatectomy was performed, followed by STZ administration to induce diabetes and destroy any remaining endogenous islets. Application of the autologous islets to the momentum using PPCN restored normoglycemia with minimal insulin requirements for over 100 days. These results support the use of PPCN as a scaffold for minimally invasive delivery of islets to the omentum of pancreatitis patients and highlight the importance of scaffold antioxidant properties as a new mechanism to protect islet function and maximize long-term autologous graft performance. One Sentence SummaryOmentum islet transplantation using a thermoresponsive, antioxidative polymer supports autologous islet viability and function in nonhuman primates.

bioengineering↗

bioRxiv2023

SHRIMP: Genetically Encoded mScarlet-derived Red Fluorescent Hydrogen Peroxide Sensor with High Brightness and Minimal Photoactivation

Red fluorescent protein (RFP) derived indicators are popular due to advantages such as increased imaging depth and reduced autofluorescence and cytotoxicity. However, most RFP-based indicators have low brightness and are susceptible to blue-light-induced photoactivation. In this study, we aimed to overcome the limitations of existing red fluorescent indicators. We utilized mScarlet-I, a highly bright and robust monomeric RFP, to develop a circularly permuted variant called cpmScarlet. We further engineered cpmScarlet into a novel red fluorescent indicator specifically for hydrogen peroxide (H2O2), a crucial reactive oxygen species (ROS) involved in redox signaling and oxidative stress. The resultant indicator, SHRIMP (mScarlet-derived H2O2 Redox Indicator with Minimal Photoactivation), exhibited excitation and emission peaks at [~]570 and 595 nm, respectively, and demonstrated a maximum five-fold fluorescence turn-off response to H2O2. Importantly, SHRIMP was not susceptible to blue-light-induced photoactivation and showed high brightness both in its purified protein form and when expressed in mammalian cells. We successfully employed SHRIMP to visualize H2O2 dynamics in mammalian cells with exogenously added H2O2 and in activated macrophages. Additionally, we demonstrated its utility for multiparameter imaging by co-expressing SHRIMP with GCaMP6m, a green fluorescent calcium indicator, enabling simultaneous monitoring of H2O2 and calcium dynamics in mammalian cells in response to thapsigargin (TG) and epidermal growth factor (EGF) stimulation. Furthermore, we expressed SHRIMP in isolated primary mouse islet tissue, and SHRIMP exhibited excellent brightness and capability for effective detection of H2O2 production during streptozotocin (STZ)-induced {beta}-cell damage. This study successfully transformed mScarlet-I, a bright and robust monomeric RFP, into a circularly permuted variant (cpmScarlet) and developed the first cpmScarlet-based genetically encoded fluorescent indicator called SHRIMP. SHRIMP exhibits high brightness and insensitivity to photoactivation and is a valuable tool for real-time monitoring of H2O2 dynamics in various biological systems. Further research may yield an expanded family of cpmScarlet-based red fluorescent indicators with enhanced photophysical properties.

biochemistry↗

bioRxiv2021

Voxelated Bioprinting of Mechanically Robust Multiscale Porous Scaffolds for Pancreatic Islets

Three-dimensional (3D) bioprinting additively assembles bio-inks to manufacture tissue-mimicking biological constructs, but with the typical building blocks limited to one-dimensional filaments. Here, we develop a technique for the digital assembly of spherical particles (DASP), which are effectively zero-dimensional voxels - the basic unit of 3D structures. We show that DASP enables on-demand generation, deposition, and assembly of viscoelastic bio-ink droplets. We establish a phase diagram that outlines the viscoelasticity of bio-inks required for printing spherical particles of good fidelity. Moreover, we develop a strategy for engineering bio-inks with independently controllable viscoelasticity and mesh size. Using DASP, we create mechanically robust, multiscale porous scaffolds composed of interconnected yet distinguishable hydrogel particles. Finally, we demonstrate the application of the scaffolds in encapsulating human pancreatic islets for responsive insulin release. Together with the knowledge of bio-ink design, DASP might be used to engineer highly heterogeneous yet tightly organized tissue constructs for therapeutic applications. Table of ContentsA three-dimensional bioprinting technology is developed to enable on-demand generation, deposition, and assembly of viscoelastic bio-ink droplets in a biofriendly environment. A strategy is developed to independently control elasticity, viscosity, and mesh size of bio-inks. The technique allows for easy manufacturing mechanically robust multiscale porous scaffolds, which can be used to encapsulate human pancreatic islets for sustained responsive insulin release. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=182 SRC="FIGDIR/small/449587v1_ufig1.gif" ALT="Figure 1"> View larger version (58K): org.highwire.dtl.DTLVardef@1c0f2eaorg.highwire.dtl.DTLVardef@8bf924org.highwire.dtl.DTLVardef@191bd3aorg.highwire.dtl.DTLVardef@11e5ce1_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2021

Human stem cell model of HNF1A deficiency shows uncoupled insulin to C-peptide secretion with accumulation of abnormal insulin granules

Mutations in HNF1A cause Maturity Onset Diabetes of the Young type 3 (MODY3), the most prevalent form of monogenic diabetes. We generated stem cell-derived pancreatic endocrine cells from human embryonic stem cells (hESCs) with induced hypomorphic mutations in HNF1A. Using these cells, we show that HNF1A orchestrates a transcriptional program required for distinct aspects of {beta}-cell fate and function. During islet cell differentiation, HNF1A deficiency biases islet endocrine cells towards an -cell fate associated with PAX4 down-regulation. HNF1A- deficient {beta}-cells display impaired basal and glucose stimulated-insulin secretion in association with a reduction in CACNA1A and intracellular calcium levels, and impaired insulin granule exocytosis in association with SYT13 down-regulation. Knockout of PAX4, CACNA1A and SYT13 reproduce the relevant phenotypes. Reduction of insulin secretion is associated with accumulation of enlarged secretory granules, and altered stoichiometry of secreted insulin to C-peptide. In HNF1A deficient {beta}-cells, glibenclamide, a sulfonylurea drug used in the treatment of MODY3 patients, increases intracellular calcium to levels beyond those achieved by glucose, and restores C-peptide and insulin secretion to a normal stoichiometric ratio. To study HNF1A deficiency in the context of a human disease model, we also generated stem cell-derived pancreatic endocrine cells from two MODY3 patients induced pluripotent stem cells (iPSCs). While insulin secretion defects are constitutive in cells with complete HNF1A loss of function, {beta}-cells heterozygous for hypomorphic HNF1A mutations are initially normal, but lose the ability to secrete insulin and acquire abnormal stoichiometric secretion ratios. Importantly, the defects observed in these stem cell models are also seen in circulating proportions of insulin:C-peptide in nine MODY3 patients. One sentence of summaryDeficiency of the transcription factor HNF1A biases islet endocrine cell fate towards -cells, impairs intracellular calcium homeostasis and insulin exocytosis, alters the stoichiometry of insulin to C-peptide release, and leads to an accumulation of abnormal insulin secretory granules in {beta}-cells.

cell biology↗

bioRxiv2020

Establishing cell-intrinsic limitations in cell cycle progression controls graft growth and promotes differentiation of pancreatic endocrine cells

Limitations in cell proliferation are a key barrier to reprogramming differentiated cells to pluripotent stem cells, and conversely, acquiring these limitations may be important to establish the differentiated state. The pancreas, and beta cells in particular have a low proliferative potential, which limits regeneration, but how these limitations are established is largely unknown. Understanding proliferation potential is important for the safty of cell replacement therapy with cell products made from pluripotent stem cell which have unlimited proliferative potential. Here we test a novel hypothesis, that these limitations are established through limitations in S-phase progression. We used a stem cell-based system to expose differentiating stem cells to small molecules that interfere with cell cycle progression either by inducing G1 arrest, impairing S-phase entry, or S-phase completion. Upon release from these molecules, we determined growth potential, differentiation and function of insulin-producing endocrine cells both in vitro and after grafting in vivo. We found that the combination of G1 arrest with a compromised ability to complete DNA replication promoted the differentiation of pancreatic progenitor cells towards insulin-producing cells, improved the stability of the differentiated state, and protected mice from diabetes without the formation of cystic growths. Therefore, a compromised ability to enter S-phase and replicate the genome is a functionally important property of pancreatic endocrine differentiation, and can be exploited to generate insulin-producing organoids with predictable growth potential after transplantation.

cell biology↗