Biology subjects

Saunders, D. C.

Publications and source records attributed to Saunders, D. C..

9 recordsLinked to original sources

bioRxiv2026

AAV-DJ Enables Targeted Gene Modulation in Human Ovarian Cells

Study question: Can adeno-associated viruses (AAVs) efficiently and safely transduce human primary granulosa cells and human ovarian stromal fibroblasts to modulate the expression of genes involved in follicle recruitment and ovarian microenvironment regulation? Summary answer: AAVs can efficiently transduce human primary granulosa cells and human ovarian stromal cells with low toxicity, with the serotype DJ (AAV-DJ) demonstrating superior performance. AAVs enable targeted modulation of ovarian genes such as AMHR2 in human primary granulosa cells and TGF{beta}1 in human ovarian stromal cells, supporting the feasibility of gene-based approaches to improve ovarian function. What is known already: Infertility affects approximately 15% of couples, with diminished oocyte quality and quantity being major contributors, particularly with advanced reproductive age. Assisted reproductive technologies rely primarily on hormonal stimulation and do not directly target molecular pathways governing folliculogenesis or the ovarian microenvironment. Animal studies suggest that gene therapy using AAVs offers a promising strategy for tissue-specific and durable gene modulation, yet its application in human ovarian cells remains largely unexplored. Study design, size, duration: This was an experimental laboratory study using primary human granulosa cells from patients undergoing in vitro fertilization (IVF) and ovarian stromal cells obtained from post-pubertal patients who donated surplus ovarian tissue from ovarian tissue cryopreservation procedures for research. Sixteen AAV serotypes were evaluated for transduction efficiency, toxicity, and expression dynamics, followed by functional gene knockdown studies using AAV-DJ-based shRNA vectors. Participants/materials, setting, methods: Primary human granulosa cells and ovarian stromal cells were isolated from follicular aspirates and ovarian tissue, respectively. Cells were transduced with GFP-expressing AAV serotypes at varying multiplicities of infection (MOIs), incubation times, and culture days to assess transduction efficiency and cytotoxicity. Lead serotypes were further evaluated in dose-response studies. Functional gene modulation was assessed using AAV-DJ-mediated shRNA knockdown of AMHR2 in granulosa cells and TGF{beta}1 in stromal cells, quantified by RT-qPCR, immunofluorescence, and western blotting. Main results and the role of chance: Six of sixteen AAV serotypes (AAV2, AAV3, AAV6, AAV9, AAV-DJ, and AAV-X1.1) achieved greater than 10% transduction efficiency in human primary granulosa cells. Among these, AAV-DJ demonstrated the highest efficiency across all MOIs tested, reaching up to 48% transduction at 100,000 MOI, significantly outperforming AAV2 and AAV9. Toxicity increased with viral dose for all serotypes but remained comparable across vectors, with AAV-DJ exhibiting higher efficiency with comparable toxicity. The AAV-DJ-shRNA-AMHR2 construct produced a significant reduction in expression of AMHR2 mRNA by ~81% (p<0.05) in human primary granulosa cells as well as at the protein level in HEK293T-AMHR2-ZsGreen engineered cells (~85% knockdown, p<0.01). In human ovarian stromal cells, AAV-DJ preferentially transduced EMILIN1 and ACTA2-positive fibroblast and myofibroblast-like cells and enabled robust knockdown of TGF{beta}1 at both transcript (~89% knockdown, p<0.05) and protein levels (~67% knockdown, p<0.0001) using AAV-DJ-shRNA-TGF{beta}1 construct. Limitations, reasons for caution: These experiments were performed in vitro using primary human ovarian cells, which may not fully recapitulate the in vivo ovarian environment. Functional reproductive outcomes were not assessed, and future studies will be required to evaluate the effects of gene modulation on folliculogenesis and fertility. Wider implications of the findings: These findings establish AAV-DJ as a promising vector for targeted gene modulation in human ovarian cells. This approach introduces a novel framework for addressing infertility and ovarian function by directly targeting molecular pathways involved in follicle recruitment and ovarian stromal signaling, potentially enhancing current assisted reproductive technologies.

developmental biology↗

bioRxiv2024

Mapping histological and functional maturation of human endocrine pancreas across early postnatal periods

Human endocrine cell differentiation and islet morphogenesis play critical roles in determining islet cell mass and function, but the events and timeline of these processes are incompletely defined. To better understand early human islet cell development and maturation, we collected 115 pediatric pancreata and mapped morphological and spatiotemporal changes from birth through the first ten years of life. Using quantitative analyses and a combination of complementary tissue imaging approaches, including confocal microscopy and whole-slide imaging, we developed an integrated model for endocrine cell formation and islet architecture, including endocrine cell type heterogeneity and abundance, endocrine cell proliferation, and islet vascularization and innervation. We also assessed insulin and glucagon secretory profiles in isolated islet preparations from pediatric donors aged 2 months to 10 years and found a temporal difference in the maturation of insulin secretion compared to glucagon secretion. This comprehensive summary of postnatal and pediatric pancreatic islet development provides a framework for future studies and integration of emerging genetic and genomic data related to islet biology and diabetes risk.

developmental biology↗

bioRxiv2024

Heterogeneous endocrine cell composition defines human islet functional phenotypes

Phenotyping and genotyping initiatives within the Integrated Islet Distribution Program (IIDP), the largest source of human islets for research in the U.S., provide standardized assessment of islet preparations distributed to researchers, enabling the integration of multiple data types. Data from islets of the first 299 organ donors without diabetes, analyzed using this pipeline, highlights substantial heterogeneity in islet cell composition associated with hormone secretory traits, sex, reported race and ethnicity, genetically predicted ancestry, and genetic risk for type 2 diabetes (T2D). While and {beta} cell composition influenced insulin and glucagon secretory traits, the abundance of {delta} cells showed the strongest association with insulin secretion and was also associated with the genetic risk score (GRS) for T2D. These findings have important implications for understanding mechanisms underlying diabetes heterogeneity and islet dysfunction and may provide insight into strategies for personalized medicine and {beta} cell replacement therapy.

physiology↗

bioRxiv2021

RFX6-mediated dysregulation defines human β cell dysfunction in early type 2 diabetes

A hallmark of type 2 diabetes (T2D), a major cause of world-wide morbidity and mortality, is dysfunction of insulin-producing pancreatic islet {beta} cells1-3. T2D genome-wide association studies (GWAS) have identified hundreds of signals, mostly in the non-coding genome and overlapping {beta} cell regulatory elements, but translating these into biological mechanisms has been challenging4-6. To identify early disease-driving events, we performed single cell spatial proteomics, sorted cell transcriptomics, and assessed islet physiology on pancreatic tissue from short-duration T2D and control donors. Here, through integrative analyses of these diverse modalities, we show that multiple gene regulatory modules are associated with early-stage T2D {beta} cell-intrinsic defects. One notable example is the transcription factor RFX6, which we show is a highly connected {beta} cell hub gene that is reduced in T2D and governs a gene regulatory network associated with insulin secretion defects and T2D GWAS variants. We validated the critical role of RFX6 in {beta} cells through direct perturbation in primary human islets followed by physiological and single nucleus multiome profiling, which showed reduced dynamic insulin secretion and large-scale changes in the {beta} cell transcriptome and chromatin accessibility landscape. Understanding the molecular mechanisms of complex, systemic diseases necessitates integration of signals from multiple molecules, cells, organs, and individuals and thus we anticipate this approach will be a useful template to identify and validate key regulatory networks and master hub genes for other diseases or traits with GWAS data.

cell biology↗

bioRxiv2021

Combinatorial transcription factor profiles predict mature and functional human islet α and β cells

Islet-enriched transcription factors (TFs) exert broad control over cellular processes in pancreatic and {beta} cells and changes in their expression are associated with developmental state and diabetes. However, the implications of heterogeneity in TF expression across islet cell populations are not well understood. To define this TF heterogeneity and its consequences for cellular function, we profiled >40,000 cells from normal human islets by scRNA-seq and stratified and {beta} cells based on combinatorial TF expression. Subpopulations of islet cells co-expressing ARX/MAFB ( cells) and MAFA/MAFB ({beta} cells) exhibited greater expression of key genes related to glucose sensing and hormone secretion relative to subpopulations expressing only one or neither TF. Moreover, all subpopulations were identified in native pancreatic tissue from multiple donors. By Patch-seq, MAFA/MAFB co-expressing {beta} cells showed enhanced electrophysiological activity. Thus, these results indicate combinatorial TF expression in islet and {beta} cells predicts highly functional, mature subpopulations.

genomics↗

bioRxiv2020

SARS-CoV-2 Cell Entry Factors ACE2 and TMPRSS2 are Expressed in the Pancreas but Not in Islet Endocrine Cells

Summary/AbstractReports of new-onset diabetes and diabetic ketoacidosis in individuals with COVID-19 have led to the hypothesis that SARS-CoV-2, the virus that causes COVID-19, is directly cytotoxic to pancreatic islet {beta} cells. This would require binding and entry of SARS-CoV-2 into host {beta} cells via cell surface co-expression of ACE2 and TMPRSS2, the putative receptor and effector protease, respectively. To define ACE2 and TMPRSS2 expression in the human pancreas, we examined six transcriptional datasets from primary human islet cells and assessed protein expression by immunofluorescence in pancreata from donors with and without diabetes. ACE2 and TMPRSS2 transcripts were low or undetectable in pancreatic islet endocrine cells as determined by bulk or single cell RNA sequencing, and neither protein was detected in or {beta} cells from these donors. Instead, ACE2 protein was expressed in the islet and exocrine tissue microvasculature and also found in a subset of pancreatic ducts, whereas TMPRSS2 protein was restricted to ductal cells. The absence of significant ACE2 and TMPRSS2 co-expression in islet endocrine cells reduces the likelihood that SARS-CoV-2 directly infects pancreatic islet {beta} cells through these cell entry proteins.

cell biology↗

bioRxiv2020

Coordinated interactions between endothelial cells and macrophages in the islet microenvironment promote beta cell regeneration

Endogenous {beta} cell regeneration could alleviate diabetes, but proliferative stimuli within the islet microenvironment are incompletely understood. We previously found that {beta} cell recovery following hypervascularization-induced {beta} cell loss involves interactions with endothelial cells (ECs) and macrophages (M{Phi}s). Here we show that proliferative ECs modulate M{Phi} infiltration and phenotype during {beta} cell loss, and recruited M{Phi}s are essential for {beta} cell recovery. Furthermore, VEGFR2 inactivation in quiescent ECs accelerates islet vascular regression during {beta} cell recovery and leads to increased {beta} cell proliferation without changes in M{Phi} phenotype or number. Transcriptome analysis of {beta} cells, ECs, and M{Phi}s reveals that {beta} cell proliferation coincides with elevated expression of extracellular matrix remodeling molecules and growth factors likely driving activation of proliferative signaling pathways in {beta} cells. Collectively, these findings suggest a new {beta} cell regeneration paradigm whereby coordinated interactions between intra-islet M{Phi}s, ECs, and extracellular matrix mediate {beta} cell self-renewal.

cell biology↗

bioRxiv2020

Pancreatlas: a comprehensive and interactive image atlas of the human pancreas in health and disease

Human tissue phenotyping generates complex spatial information from numerous imaging modalities, yet images typically become static figures for publication and original data and metadata are rarely available. While comprehensive image maps exist for some organs, most resources have limited support for multiplexed imaging or have non-intuitive user interfaces. Therefore, we built a Pancreatlas resource that integrates several technologies into a novel interface, allowing users to access richly annotated web pages, drill down to individual images, and deeply explore data online. The current version of Pancreatlas contains over 800 unique images acquired by whole-slide scanning, confocal microscopy, and imaging mass cytometry, and is available at https://www.pancreatlas.org. To create this human pancreas-specific biological imaging resource, we developed a React-based web application and Python-based application programming interface, collectively called Flexible Framework for Integrating and Navigating Data (FFIND), which can be adapted beyond Pancreatlas to meet countless imaging or other structured data management needs.

cell biology↗

bioRxiv2019

Human pseudoislet system enables detection of differences in G-protein-coupled-receptor signaling pathways between α and β cells

G-protein-coupled-receptors (GPCRs) modulate insulin secretion from {beta} cells and glucagon secretion from cells. Here, we developed an integrated approach to study the function of primary human islet cells using genetically modified pseudoislets that resemble native islets across multiple parameters. We studied the Gi and Gq GPCR pathways by expressing the designer receptors exclusively activated by designer drugs (DREADDs) hM4Di or hM3Dq. Activation of Gi signaling reduced insulin and glucagon secretion, while activation of Gq signaling stimulated glucagon secretion but had both stimulatory and inhibitory effects on insulin secretion. Further, we developed a microperifusion system that allowed synchronous acquisition of GCaMP6f biosensor signal and hormone secretory profiles and showed that the dual effects for Gq signaling occur through changes in intracellular Ca2+. By combining pseudoislets with a microfluidic system, we co-registered intracellular signaling dynamics and hormone secretion and demonstrated differences in GPCR signaling pathways between human {beta} and cells.

cell biology↗