ABSTRACT The biological mechanisms underlying arteriovenous fistula (AVF) maturation in hemodialysis patients remain poorly understood despite decades of research. To address this gap, we investigated the cellular changes in the venous wall after fistula creation in histological biopsies of longitudinal veins and AVF samples (N=23 patients). Using single-cell RNA sequencing of 70,281 cells from pre-access veins, mature, and failed AVFs (N=20 patients), we created a complementary transcriptomic atlas of the human vein before and after anastomosis. Postoperatively, the fistula exhibited increased intimal hyperplasia and cell number but reduced cell density, indicating that extracellular matrix (ECM) deposition was more prominent than cell accumulation. Analysis of 14,475 cells from fistulas obtained within one week of creation revealed that inflammation drives early adaptation across all vascular cell types. This includes the pro-inflammatory activation of endothelial cells (ECs) and production of a hyaluronic acid-rich neointima by fibroblasts. By 13 ± 6 weeks, transcriptomic profiles continue to reflect active healing of the vasculature by ECM-producing myofibroblasts and fibroblasts that were found localized throughout the vascular wall, including the intima, using immunofluorescence and in-situ hybridization. Postoperative ECs maintained significant hemostatic adaptations and upregulation of inflammatory molecules ( ACKR3, ICAM1, IL1R1, COL8A1 ) supporting their role as gatekeepers of immune cell infiltration. Comparative analyses of failed versus mature AVFs revealed persistent inflammatory signaling among macrophages, ECs, myofibroblasts, and fibroblasts in association with AVF failure. These findings uncover previously unrecognized cellular and molecular patterns in human veins following AVF creation, providing novel insights and potential therapeutic targets to improve AVF outcomes. TRANSLATIONAL STATEMENT Arteriovenous fistulas (AVF) are a special type of blood vessels that provides access to a patient’s bloodstream during hemodialysis treatments. The AVF is surgically created by connecting an artery and a vein, after which the vein heals and enlarges in a process called “maturation”. We do not fully understand how maturation occurs. This prevents us from designing therapies that ensure proper enlargement of the AVF, which fails in up to 40% of patients. This study investigates how a vein transforms into an AVF in 43 patients with end-stage kidney disease undergoing surgery for AVF creation. We analyze the modifications of structural components of the vein and of diverse populations of cells that direct healing and maturation. Our findings suggest that, contrary to current beliefs, the best therapies to improve AVF maturation should target the accumulation of non-cellular components in the vein and the inflammatory factors that trigger such accumulation.

Human Cytomegalovirus (HCMV) infection can result in either productive or latent infection, the latter being the basis for the virus life-long persistence. Intriguingly, monocytes, which support latent infection, become permissive to productive infection upon differentiation to macrophages. However, the molecular factors explaining these differentiation-driven differences are not fully understood and have been so far attributed to chromatin-mediated repression of the viral genome. Here, by using metabolic labeling of newly synthesized RNA early in monocyte and macrophage infection, we discover a major early block in viral gene expression, and viral transcripts are barely detected in infected monocytes. By unbiasedly analyzing the changes between monocytes and their differentiated counterparts, we reveal that the levels of several cell surface proteins involved in HCMV entry are upregulated upon monocyte to macrophage differentiation, and correspondingly we uncover HCMV entry into monocytes compared to macrophages is extremely inefficient. Remarkably, ectopic expression of a canonical HCMV entry receptor in monocytes facilitates productive infection of these cells, demonstrating that given efficient viral entry, monocytes, like macrophages, have the capacity to support productive infection. Among the cell surface proteins that are upregulated upon monocyte differentiation are several integrins, which we show play an important role in HCMV entry into macrophages, partially explaining the differences in viral entry. Overall, our findings reveal that a previously unrecognized major barrier for productive infection in monocytes is entry, adding a critical layer to the paradigm of HCMV latency.

Two-dimensional neuronal cultures have a limited ability to recapitulate the in vivo environment of the brain. Here, we introduce a three-dimensional in vitro model for human glia-to-neuron conversion, surpassing the spatial and temporal constrains of two-dimensional cultures. Focused on direct conversion to induced dopamine neurons (iDANs) relevant to Parkinson disease, the model generates functionally mature iDANs in 2 weeks and allows long-term survival. As proof of concept, we use single-nucleus RNA sequencing and molecular lineage tracing during iDAN generation and find that all glial subtypes generate neurons and that conversion relies on the coordinated expression of three neural conversion factors. We also show the formation of mature and functional iDANs over time. The model facilitates molecular investigations of the conversion process to enhance understanding of conversion outcomes and offers a system for in vitro reprogramming studies aimed at advancing alternative therapeutic strategies in the diseased brain.
Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.

Direct neural conversion of endogenous non-neuronal cells, such as resident glia, into therapeutic neurons has emerged as a promising strategy for brain repair, aiming to restore lost or damaged neurons. Proof-of-concept has been obtained from animal studies, yet these models do not efficiently recapitulate the complexity of the human brain, and further refinement is necessary before clinical translation becomes viable. One important aspect is the need to achieve efficient and precise targeting of human glial cells using non-integrating viral vectors that exhibit a high degree of cell type specificity. While various naturally occurring or engineered adeno-associated virus (AAV) serotypes have been utilized to transduce glia, efficient targeting of human glial cell types remains an unsolved challenge. In this study, we employ AAV capsid library engineering to find AAV capsids that selectively target human glia in vitro and in vivo. We have identified two families of AAV capsids that induce efficient targeting of human glia both in glial spheroids and after glial progenitor cell transplantation into the rat forebrain. Furthermore, we show the robustness of this targeting by transferring the capsid peptide from the parent AAV2 serotype onto the AAV9 serotype, which facilitates future scalability for the larger human brain.
Copyright © 2024 Giacomoni, Åkerblom, Habekost, Fiorenzano, Kajtez, Davidsson, Parmar and Björklund.

To better understand sodium channel (SCN5A)-related cardiomyopathies, we generated ventricular cardiomyocytes from induced pluripotent stem cells obtained from a dilated cardiomyopathy patient harbouring the R222Q mutation, which is only expressed in adult SCN5A isoforms. Because the adult SCN5A isoform was poorly expressed, without functional differences between R222Q and control in both embryoid bodies and cell sheet preparations (cultured for 29-35 days), we created heart-on-a-chip biowires which promote myocardial maturation. Indeed, biowires expressed primarily adult SCN5A with R222Q preparations displaying (arrhythmogenic) short action potentials, altered Na+ channel biophysical properties and lower contractility compared to corrected controls. Comprehensive RNA sequencing revealed differential gene regulation between R222Q and control biowires in cellular pathways related to sarcoplasmic reticulum and dystroglycan complex as well as biological processes related to calcium ion regulation and action potential. Additionally, R222Q biowires had marked reductions in actin expression accompanied by profound sarcoplasmic disarray, without differences in cell composition (fibroblast, endothelial cells, and cardiomyocytes) compared to corrected biowires. In conclusion, we demonstrate that in addition to altering cardiac electrophysiology and Na+ current, the R222Q mutation also causes profound sarcomere disruptions and mechanical destabilization. Possible mechanisms for these observations are discussed.
Copyright © 2023 Elsevier Ltd. All rights reserved.