The underlying mechanisms of atherosclerosis, the second leading cause of death among Werner syndrome (WS) patients, are not fully understood. Here, we establish an in vitro co-culture system using macrophages (iMφs), vascular endothelial cells (iVECs), and vascular smooth muscle cells (iVSMCs) derived from induced pluripotent stem cells. In co-culture, WS-iMφs induces endothelial dysfunction in WS-iVECs and characteristics of the synthetic phenotype in WS-iVSMCs. Transcriptomics and open chromatin analysis reveal accelerated activation of type I interferon signaling and reduced chromatin accessibility of several transcriptional binding sites required for cellular homeostasis in WS-iMφs. Furthermore, the H3K9me3 levels show an inverse correlation with retrotransposable elements, and retrotransposable element-derived double-stranded RNA activates the DExH-box helicase 58 (DHX58)-dependent cytoplasmic RNA sensing pathway in WS-iMφs. Conversely, silencing type I interferon signaling in WS-iMφs rescues cell proliferation and suppresses cellular senescence and inflammation. These findings suggest that Mφ-specific inhibition of type I interferon signaling could be targeted to treat atherosclerosis in WS patients.
© 2024. The Author(s).

In vitro maturation (IVM) is an infertility treatment used during in vitro fertilization (IVF) procedures in which immature oocytes are matured outside the body, limiting the excessive hormone doses required for retrieval of ready-to-fertilize oocytes. To overcome the historically low embryo formation rate associated with IVM, we have recently demonstrated that co-culture of hiPSC-derived ovarian support cells (OSCs) yielded higher rates of oocyte maturation and euploid embryo formation, by mimicking the complex ovarian environment in vitro, offering a novel solution to overcome the IVM main limitation. To translate this process into clinics, we sourced and engineered a compliant female clinical-grade (CG) hiPSC line to derive OSCs with similar quality attributes and clinical outcomes to results previously demonstrated with a research hiPSC line. We further optimized our manufacturing protocols to enable increased scale and substituted reagents with appropriate higher-quality alternatives. This strategic approach to product development has successfully met scalable manufacturing needs and ultimately resulted in a product of improved reproducibility, purity, and efficacy. Our findings support the use of a similar strategy to fine-tune hiPSC-derived products facilitating translation to clinical applications.

p97/VCP, a hexametric member of the AAA-ATPase superfamily, has been associated with a wide range of cellular protein pathways, such as proteasomal degradation, the unfolding of polyubiquitinated proteins, and autophagosome maturation. Autosomal dominant p97/VCP mutations cause a rare hereditary multisystem disorder called IBMPFD/ALS (Inclusion Body Myopathy with Paget's Disease and Frontotemporal Dementia/Amyotrophic Lateral Sclerosis), characterized by progressive weakness and subsequent atrophy of skeletal muscles, and impacting bones and brains, such as Parkinson's disease, Lewy body disease, Huntington's disease, and amyotrophic lateral ALS. Among all disease-causing mutations, Arginine 155 to Histidine (R155H/+) was reported to be the most common one, affecting over 50% of IBMPFD patients, resulting in disabling muscle weakness, which might eventually be life-threatening due to cardiac and respiratory muscle involvement. Induced pluripotent stem cells (iPSCs) offer an unlimited resource of cells to study pathology's underlying molecular mechanism, perform drug screening, and investigate regeneration. Using R155H/+ patients' fibroblasts, we generated IPS cells and corrected the mutation (Histidine to Arginine, H155R) to generate isogenic control cells before differentiating them into myotubes. The further proteomic analysis allowed us to identify differentially expressed proteins associated with the R155H mutation. Our results showed that R155H/+ cells were associated with dysregulated expression of several proteins involved in skeletal muscle function, cytoskeleton organization, cell signaling, intracellular organelles organization and function, cell junction, and cell adhesion. Our findings provide molecular evidence of dysfunctional protein expression in R155H/+ myotubes and offer new therapeutic targets for treating IBMPFD/ALS.
Copyright © 2023 Luzzi, Wang, Li, Iacovino and Chou.

Mutations in ARID1B , a member of the mSWI/SNF complex, cause severe neurodevelopmental phenotypes with elusive mechanisms in humans. The most common structural abnormality in the brain of ARID1B patients is agenesis of the corpus callosum (ACC). This condition is characterized by a partial or complete absence of the corpus callosum (CC), an interhemispheric white matter tract that connects distant cortical regions. Using human neural organoids, we identify a vulnerability of callosal projection neurons (CPNs) to ARID1B haploinsufficiency, resulting in abnormal maturation trajectories and dysregulation of transcriptional programs of CC development. Through a novel in vitro model of the CC tract, we demonstrate that ARID1B mutations reduce the proportion of CPNs capable of forming long-range projections, leading to structural underconnectivity phenotypes. Our study uncovers new functions of the mSWI/SNF during human corticogenesis, identifying cell-autonomous defects in axonogenesis as a cause of ACC in ARID1B patients. Abstract Figure Graphical abstract Human callosal projection neurons are vulnerable to ARID1B haploinsufficiency. (Top) During healthy development, callosal projection neurons (CPNs) project long interhemispheric axons, forming the corpus callosum (CC) tract, which can be modeled in vitro . (Bottom) In ARID1B patients, transcriptional dysregulation of genetic programs of CC development reduces the formation of long-range projections from CPNs, causing CC agenesis in vivo and underconnectivity phenotypes in vitro .

Advances in cellular engineering, as well as gene, and cell therapy, may be used to produce human tissues with programmable genetically enhanced functions designed to model and/or treat specific diseases. Fabrication of synthetic human liver tissue with these programmable functions has not been described. By generating human iPSCs with target gene expression controlled by a guide RNA-directed CRISPR-Cas9 synergistic-activation-mediator, we produced synthetic human liver tissues with programmable functions. Such iPSCs were guide-RNA-treated to enhance expression of the clinically relevant CYP3A4 and UGT1A1 genes, and after hepatocyte-directed differentiation, cells demonstrated enhanced functions compared to those found in primary human hepatocytes. We then generated human liver tissue with these synthetic human iPSC-derived hepatocytes (iHeps) and other non-parenchymal cells demonstrating advanced programmable functions. Fabrication of synthetic human liver tissue with modifiable functional genetic programs may be a useful tool for drug discovery, investigating biology, and potentially creating bioengineered organs with specialized functions.
© 2022 The Authors.