Human induced pluripotent stem cells (hiPSCs) show great promise in the development of novel strategies to mitigate reproductive diseases and promote successful reproductive outcomes. Recently, a novel approach for the fast and efficient differentiation of ovarian support cells (OSCs) to generate a versatile platform for basic research and clinical applications was demonstrated. This study details the clinical process development and application of an OSC product, known as Fertilo , to improve the in vitro maturation (IVM) of human oocytes, a method referred to as OSC-IVM. First, transcription factor (TF) mediated OSC differentiation using research-grade raw materials was shown to produce granulosa-like cells that improve the MII maturation rate of human oocytes. To support clinical application, several raw material upgrades were initiated, including substitution of the differentiation matrix with a higher-quality alternative, laminin-521, and the generation of a clinically suitable hiPSC seed bank and master cell bank. Single cell RNA sequencing of OSCs generated using the updated protocol for clinical translation demonstrated the consistency and reproducibility of cellular outcomes. Next, analytical release testing of the clinical product was performed and a murine oocyte maturation assay was developed to establish the potency of OSCs for use in OSC-IVM. Finally, the qualified Fertilo product was applied in a two-phase longitudinal cohort analysis, with the results showing improvement in key outcomes compared to traditional IVM treatment. Our findings demonstrate the first-time clinical development and application of an hiPSC-derived product to improve reproductive outcomes after IVM and advance women’s health.

Hepatitis B virus (HBV)-DNA integration into the host genome contributes to hepatocellular carcinoma (HCC) development. KMT2B is the second most frequent locus of HBV-DNA integration in HCC; however, its role and function remain unclear. We aimed to clarify the impact of HBV-KMT2B integration in HCC development using a human genome-edited induced pluripotent stem cell (iPSCs) model.
Based on the genetic information on HBV-KMT2B integration in HCC, we determined its complete DNA sequence and transcript variants. To exclude the effect of other oncogenic mutations, we reproduced HBV integration in healthy donor iPSCs with an intact genome and analyzed its effects using iPSC-derived hepatic progenitor cells (HPCs) and hepatocytes (iPS-Heps).
The reproduced HBV-KMT2B integration significantly upregulated the proliferation of hepatic cells. Comprehensive transcriptional and epigenetic analyses revealed enhanced expression of cell cycle-related genes in hepatic cells with HBV-KMT2B integration based on perturbation of histone 3 lysine 4 tri-methylation (H3K4me3), mimicking that in the original HCC sample. Long-read RNA-sequence detected the common KMT2B transcript variants in the HCC sample and HPCs. Overexpression of the truncated variant significantly enhanced proliferation of hepatic cells, whereas HBV-KMT2B fusion transcripts did not enhance proliferation. HBV-KMT2B-integrated HPCs exhibited replication stress and DNA damage, indicating that our model initiated the process of hepatocarcinogenesis due to abnormally promoted KMT2B function.
Our disease model using genetically engineered iPSCs provides the first insight into both the KMT2B function in HCC development and the oncogenic processes by HBV-KMT2B integration. We clarified the novel oncogenic mechanism in HBV-related HCC due to aberrant KMT2B function.
Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.

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.