Biology subjects

Millman, J. R.

Publications and source records attributed to Millman, J. R..

13 recordsLinked to original sources

bioRxiv2026

A digital twin of pancreatic islet differentiation predicts cell fate

The controlled generation of mature stem cell-derived islets (SC-islets) remains a barrier to scalable cell therapy for diabetes. Here, we develop a predictive digital model defining the cell-state-specific regulatory logic governing fate specification during human SC-islet differentiation. We integrate 400,603 cells from 9 original single-cell multiomic datasets and 52 public single-cell RNA-seq and ATAC-seq datasets across 4 cell lines and 7 differentiation protocols. This model resolves transcriptional and chromatin accessibility dynamics while enabling time-resolved inference and in silico perturbation of cell-state-specific gene regulatory networks. We identify regulators across trajectories from endoderm progenitors to pancreatic exocrine and endocrine lineages, nominating new candidate regulators. Among these candidates, we validate previously unreported roles for STAT1 as an exocrine driver and ZEB1 as a dynamic regulator of early endocrine specification and later off-target serotonergic islet cell fate. This work provides an experimentally supported predictive framework and an interactive resource comprising 1,116 simulations to prioritize transcription factors and intervention windows for refining SC-islet differentiation.

cell biology↗

bioRxiv2026

Optimizing Post-Transplantation Detection of Subcutaneously Transplanted Islets Using Dithizone Staining

IntroductionPancreatic islet transplantation is a promising therapeutic strategy to restore insulin independence in patients with type 1 diabetes mellitus. In addition to primary human islets, human pluripotent stem cell-derived islets have shown clinical promise. However, optimizing transplantation sites and improving methods for standardized islet dosing and post-transplant graft localization are key limitations. The subcutaneous space offers an alternative site for transplantation due to its accessibility and minimally invasive nature; nevertheless, poor vascularization and difficulty locating engrafted islets limit its experimental utility. The use of dithizone, a zinc-chelating dye that selectively binds the high intracellular zinc concentrations of insulin-producing {beta}-cells, may enable rapid graft localization through selective staining. Here, we investigate whether dithizone staining can reliably localize transplanted islet grafts within the subcutaneous space of murine models. MethodsSC-islets were generated from HUES8 stem cells and functionally validated using glucose-stimulated insulin secretion. Islet volume was standardized prior to transplantation using automated islet equivalent quantification with the BioRep Islet Cell Counter. Approximately 4,000 islet equivalents of either SC-islets or primary human islets were transplanted into the subcutaneous space of immunodeficient mice. Graft function was assessed longitudinally via blood glucose monitoring, glucose tolerance testing, and human C-peptide measurements. Four weeks post-transplantation, dithizone staining was applied to harvested skin tissue to localize islet grafts, and immunohistochemical validation was performed. ResultsAutomated quantification of islet equivalents enabled consistent dosing across transplantation groups. Transplanted islets maintained function in vivo, as demonstrated by reduced blood glucose levels, preserved glucose tolerance, and detectable human C-peptide. Dithizone staining produced clear and selective red labeling of insulin-producing {beta}-cell grafts within the subcutaneous tissue, enabling reliable localization and recovery of engrafted islets. Immunohistochemical analyses confirmed the presence of insulin-expressing cells at isolated islet grafts. DiscussionDithizone staining is a rapid, cost-effective approach to identify subcutaneous islet grafts and enables downstream analyses, addressing a significant limitation in islet transplantation research. When combined with standardized pre-transplant islet quantification, this approach provides a cohesive and reproducible framework for evaluating subcutaneous islet transplantation. Future studies could complement these results by assessing the potential effects of dithizone exposure and exploring its applicability across other transplantation sites and imaging modalities.

cell biology↗

bioRxiv2025

Protective IFIH1 variant reduces immune-mediated islet stress and dysfunction in a type 1 diabetes genetic background

Genome-wide association studies (GWAS) have linked dozens of genetic loci to type 1 diabetes (T1D). The IFIH1 gene, which encodes the double-stranded RNA sensor MDA5, is one such locus. The E627* single nucleotide polymorphism (SNP) in IFIH1 is associated with protection against T1D, while the A946T variant is linked to increased risk. While the E627* variant has been shown to result in a truncated protein and dampen type I interferon (IFN) signaling, its specific role in human pancreatic islet health and function remains unclear. We hypothesized that MDA5627* would protect islet cells from stress-induced dysfunction, identity loss, and cell death. Using CRISPR-Cas9 technology, we introduced the E627* and A946T variants into human pluripotent stem cells (hPSCs) derived from a T1D patient. We differentiated these hPSCs into stem cell-derived islets (SC-islets) and treated them with IFN, poly(I:C), and coxsackievirus B3, an enterovirus implicated in T1D pathogenesis. Using single-cell RNA sequencing and an array of functional assays, we investigated the variant impact on both whole SC-islets and their individual cell populations. Our analysis revealed that SC-islets, and their {beta}, , and {delta} cell subpopulations, harboring the MDA5627* variant exhibit an attenuated immune response to the various stressors compared to MDA5946T cells. We also report unique, cell-type-specific transcriptional responses that vary across variants. Notably, MDA5627* SC-islets showed reduced apoptosis rates and viral genome expression, as well as attenuated negative effects on mitochondrial function and insulin secretion in response to stress. Overall, our findings demonstrate that a clinically relevant MDA5 variant confers protection by dampening stress-mediated transcriptional responses, reducing cell dysfunction, and preventing apoptosis. These insights provide a mechanistic framework for understanding T1D pathogenesis and offer new avenues for developing preventative therapies.

cell biology↗

bioRxiv2025

Divergent Cell-Type Specific Hypoxia Responses in Human Stem Cell-Derived and Primary Islets

BackgroundThe success of stem cell-derived islet (SC-islet) therapy for type 1 diabetes is limited by poor graft survival in the hypoxic post-transplantation microenvironment. While the response of SC-islets to chronic hypoxia has been studied, a direct comparison to primary human islets during the acute hypoxic phase has not been performed. Here, we conduct a comparative single-cell transcriptomic and functional analysis of human SC-islets and primary islets exposed to acute hypoxia (1% O2) over 48 hours. ResultsOur analysis reveals two divergent response patterns. Primary islets exhibit an energy-conserving response, characterized by a {beta}-cell-specific suppression of identity genes (PDX1, MAFA) and pro-apoptotic factors like DDIT3, alongside a shift toward metabolic quiescence. In contrast, the SC-islet response is characterized by lineage instability, a significant metabolic shift toward glycolysis, and the activation of pro-apoptotic pathways. Functionally, these transcriptomic differences result in a loss of glucose-stimulated insulin secretion in both islet types, but through different mechanisms: a suppression of secretion in primary islets versus dysregulated, glucose-unresponsive insulin release in SC-islets. ConclusionThese findings demonstrate that SC-islets are particularly vulnerable under hypoxic stress, exhibiting an unstable, plastic phenotype. This comparative dataset provides a resource for developing source-specific therapeutic interventions to overcome the hypoxic barrier and improve the efficacy of cell replacement therapies.

cell biology↗

bioRxiv2024

Controlling human stem cell-derived islet composition using magnetic sorting

Stem cell-derived islets (SC-islets) consists of multiple hormone-producing cell types and offer a promising therapeutic avenue for treating type 1 diabetes (T1D). Currently, the composition of cell types generated within these SC-islets currently cannot be controlled via soluble factors during this differentiation process and consist of off-target cell types. In this study, we devised a magnetic-activated cell sorting (MACS) protocol to enrich SC-islets for CD49a, a marker associated with functional insulin-producing {beta} cells. SC-islets were generated from human pluripotent stem cells (hPSCs) using an adherent differentiation protocol and then sorted and aggregated into islet-like clusters to produce CD49a-enriched, CD49a-depleted, and unsorted SC-islets. Single-cell RNA sequencing (scRNA-seq) and immunostaining revealed that CD49a-enriched SC-islets had higher proportions of {beta} cells and improved transcriptional identity compared to other cell types. Functional assays demonstrated that CD49a-enriched SC-islets exhibited enhanced glucose-stimulated insulin secretion both in vitro and in vivo following transplantation into diabetic mice. These findings suggest that CD49a-based sorting significantly improves {beta} cell identity and the overall function of SC-islets, improving their effectiveness for T1D cell replacement therapies.

bioengineering↗

bioRxiv2024

Depolymerizing F-actin accelerates the exit from pluripotency to enhance stem cell-derived islet differentiation

In this study, we demonstrate that cytoskeletal state at the onset of directed differentiation is critical for the specification of human pluripotent stem cells (hPSCs) to all three germ layers. In particular, a polymerized actin cytoskeleton facilitates directed ectoderm differentiation, while depolymerizing F-actin promotes mesendoderm lineages. Applying this concept to a stem cell-derived islet (SC-islet) differentiation protocol, we show that depolymerizing F-actin with latrunculin A (latA) during the first 24 hours of definitive endoderm formation facilitates rapid exit from pluripotency and alters Activin/Nodal, BMP, JNK-JUN, and WNT pathway signaling dynamics. These signaling changes influence downstream patterning of the gut tube, leading to improved pancreatic progenitor identity and decreased expression of markers associated with other endodermal lineages. Continued differentiation generates islets containing a higher percentage of {beta} cells that exhibit improved maturation, insulin secretion, and ability to reverse hyperglycemia. Furthermore, this latA treatment reduces enterochromaffin cells in the final cell population and corrects differentiations from hPSC lines that otherwise fail to consistently produce pancreatic islets, highlighting the importance of cytoskeletal signaling at the onset of directed differentiation.

cell biology↗

bioRxiv2024

Modeling glioblastoma tumor progression via CRISPR-engineered brain organoids

Glioblastoma (GBM) is an aggressive form of brain cancer that is highly resistant to therapy due to significant intra-tumoral heterogeneity. The lack of robust in vitro models to study early tumor progression has hindered the development of effective therapies. Here, we develop engineered GBM organoids (eGBOs) harboring GBM subtype-specific oncogenic mutations to investigate the underlying transcriptional regulation of tumor progression. Single-cell and spatial transcriptomic analyses revealed that these mutations disrupt normal neurodevelopment gene regulatory networks resulting in changes in cellular composition and spatial organization. Upon xenotransplantation into immunodeficient mice, eGBOs form tumors that recapitulate the transcriptional and spatial landscape of human GBM samples. Integrative single-cell trajectory analysis of both eGBO-derived tumor cells and patient GBM samples revealed the dynamic gene expression changes in developmental cell states underlying tumor progression. This analysis of eGBOs provides an important validation of engineered cancer organoid models and demonstrates their utility as a model of GBM tumorigenesis for future preclinical development of therapeutics.

bioengineering↗

bioRxiv2024

Coxsackievirus B infection invokes unique cell-type specific responses in primary human pancreatic islets

Coxsackievirus B (CVB) infection has long been considered an environmental factor precipitating Type 1 diabetes (T1D), an autoimmune disease marked by loss of insulin-producing {beta} cells within pancreatic islets. Previous studies have shown CVB infection negatively impacts islet function and viability but do not report on how virus infection individually affects the multiple cell types present in human primary islets. Therefore, we hypothesized that the various islet cell populations have unique transcriptional responses to CVB infection. Here, we performed single-cell RNA sequencing on human cadaveric islets treated with either CVB or poly(I:C), a viral mimic, for 24 and 48 hours. Our global analysis reveals CVB differentially induces dynamic transcriptional changes associated with multiple cell processes and functions over time whereas poly(I:C) promotes an immune response that progressively increases with treatment duration. At the single-cell resolution, we find CVB infects all islet cell types at similar rates yet induces unique cell-type specific transcriptional responses with {beta}, , and ductal cells having the strongest response. Sequencing and functional data suggest that CVB negatively impacts mitochondrial respiration and morphology in distinct ways in {beta} and cells, while also promoting the generation of reactive oxygen species. We also observe an increase in the expression of the long-noncoding RNA MIR7-3HG in {beta} cells with high viral titers and reveal its knockdown reduces gene expression of viral proteins as well as apoptosis in stem cell-derived islets. Together, these findings demonstrate a cell-specific transcriptional, temporal, and functional response to CVB infection and provide new insights into the relationship between CVB infection and T1D.

cell biology↗

bioRxiv2023

Comparative and Integrative Single Cell Analysis Reveals New Insights into the Transcriptional Immaturity of Stem Cell-Derived β Cells

Diabetes cell replacement therapy has the potential to be transformed by human pluripotent stem cell-derived {beta} cells (SC-{beta} cells). However, the precise identity of SC-{beta} cells in relationship to primary fetal and adult {beta}-cells remains unclear. Here, we used single-cell sequencing datasets to characterize the transcriptional identity of islets from in vitro differentiation, fetal islets, and adult islets. Our analysis revealed that SC-{beta} cells share a core {beta}-cell transcriptional identity with human adult and fetal {beta}-cells, however SC-{beta} cells possess a unique transcriptional profile characterized by the persistent expression and activation of progenitor and neural-biased gene networks. These networks are present in SC-{beta} cells, irrespective of the derivation protocol used. Notably, fetal {beta}-cells also exhibit this neural signature at the transcriptional level. Our findings offer insights into the transcriptional identity of SC-{beta} cells and underscore the need for further investigation of the role of neural transcriptional networks in their development.

cell biology↗

bioRxiv2022

Multiomic profiling defines cell fate plasticity of in vitro-derived islets

Transplantation of insulin-secreting {beta}-cells differentiated from human pluripotent stem cells holds great potential as a cell therapy for treating insulin-dependent diabetes. While these stem cell-derived islets (SC-islets) are able to reverse diabetes in animal models, they are not fully equivalent to their in vivo counterparts. To better define the state of the cell types generated within these SC-islets and provide a resource for identifying deficiencies in lineage specification, we used single-cell multiomic sequencing to simultaneously measure the chromatin accessibility and transcriptional profiles of SC-islets at multiple time points as well as primary human islets. The integrated analysis of both the transcriptional and chromatin landscape for each cell provided greater resolution for defining cell identity, allowing us to derive novel gene lists for identifying each islet cell type. Furthermore, this multiomic analysis revealed that the difference between SC-{beta} cells and enterochromaffin-like cells, which are a major off-target from in vitro differentiation, is a gradient of progressive cell states rather than a stark difference in identity. The chromatin landscape of primary human islets was much more restricted, suggesting that stem cell-derived cells are not fully locked into their cell fate. While long term culture of SC-islets both in vitro and in vivo does close overall chromatin state, only in vivo transplantation directs cells toward their correct identities. Collectively, our multiomic analysis demonstrates that both the chromatin and transcriptional landscapes play significant roles in islet cell identity, and these data can be used as a resource to identify specific deficiencies in the chromatin and transcriptional state of SC-islet cell types.

cell biology↗

bioRxiv2021

Genotype-phenotype correlation analysis and therapeutic development using a patient stem cell-derived disease model of Wolfram syndrome

Wolfram syndrome is a rare genetic disorder largely caused by pathogenic variants in the WFS1 gene and manifested by diabetes mellitus, optic nerve atrophy, and progressive neurodegeneration. Recent genetic and clinical findings have revealed Wolfram syndrome as a spectrum disorder. Therefore, a genotype-phenotype correlation analysis is needed for diagnosis and therapeutic development. Here, we focus on the WFS1 c.1672C>T, p.R558C variant which is highly prevalent in the Ashkenazi-Jewish population. Clinical investigation indicates that subjects carrying the homozygous WFS1 c.1672C>T, p.R558C variant show mild forms of Wolfram syndrome phenotypes. Expression of WFS1 p.R558C is more stable compared to the other known recessive pathogenic variants associated with Wolfram syndrome. Stem cell-derived islets (SC-islets) homozygous for WFS1 c.1672C>T variant recapitulates genotype-related Wolfram phenotypes, which are milder than those of SC-islets with compound heterozygous WFS1 c.1672C>T (p.R558C), c.2654C>T (p.P885L). Enhancing residual WFS1 function by a combination treatment of chemical chaperones, sodium 4-phenylbutyrate (4-PBA) and tauroursodeoxycholic acid (TUDCA), mitigates detrimental effects caused by the WFS1 c.1672C>T, p.R558C variant and restored SC-islet function. Thus, the WFS1 c.1672C>T, p.R558C variant causes a mild form of Wolfram syndrome phenotypes, which can be remitted with a combination treatment of chemical chaperones. We demonstrate that our patient stem cell-derived disease model provides a valuable platform for further genotype-phenotype analysis and therapeutic development for Wolfram syndrome. One sentence summaryDevelopment of personalized therapy for Wolfram syndrome using genetics and iPSC model.

genetics↗

bioRxiv2019

Assessment of the in vitro function of human stem cell-derived β cells

Insulin-producing human embryonic stem cell-derived {beta} (SC-{beta}) cells are a promising cell source for diabetes cell replacement therapy. We have recently reported a differentiation strategy that produces SC-{beta} cells in islet organoids that not only undergo glucose-stimulated insulin secretion but also have an islet-like dynamic insulin release profile, displaying both first and second phase insulin secretion. The goal of this study was to further characterize the functional profile of these SC-{beta} cells in vitro. We utilized a Seahorse extracellular flux analyzer to measure mitochondrial respiration of SC-{beta} cells at low and high glucose. We also used photolithography to fabricate a microfluidic device containing microwells to immobilize SC-{beta} cells for perfusional analysis, monitoring cytoplasmic calcium using Fluo-4 AM at low and high glucose. Here we find that in addition to increased insulin secretion, SC-{beta} cells have increased cellular respiration and cytoplasmic calcium ion concentration in response to a high glucose stimulation. Our results indicate that SC-{beta} cells have similar function to that reported for islets, providing further performance characterization that could help with eventual development for diabetes cell therapy and drug screening.

bioengineering↗

bioRxiv2019

Hydrogel platform for in vitro three-dimensional assembly of human stem cell-derived β cells and endothelial cells

Differentiation of stem cells into functional replacement cells and tissues is a major goal of the regenerative medicine field. However, one limitation has been organization of differentiated cells into multi-cellular, three-dimensional assemblies. The islets of Langerhans contain many endocrine and non-endocrine cell types, such as insulin-producing {beta} cells and endothelial cells. Transplantation of exogenous islets into diabetic patients can serve as a cell replacement therapy, replacing the need for patients to inject themselves with insulin, but the number of available islets from cadaveric donors is low. We have developed a strategy of assembling human embryonic stem cell-derived {beta} cells with endothelial cells into three-dimensional aggregates on a hydrogel. The resulting islet organoids express {beta} cell markers and are functional, capable of undergoing glucose-stimulated insulin secretion. These results provide a platform for evaluating the effects of the islet tissue microenvironment on human embryonic stem cell-derived {beta} cells and other islet endocrine cells to develop tissue engineered islets.

bioengineering↗