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Despite the significant progress made in the development of epigenetic age (eAge) clocks designed to estimate the various aspects of aging, currently available models, generated using large DNA methylation microarray datasets, still cannot fully address the issues of batch effects and technical variation. This hinders the use of the publicly available eAge clocks in routine laboratory practice, and it motivates the development of cost-effective, custom epigenetic clocks that are tailored to the given biological subjects and research methods. In this study, we analyzed the local DNA methylation of mesenchymal stem cell samples during culture expansion using high-throughput targeted bisulfite sequencing (BS-seq). Using the obtained data, we trained a minimized eAge model based on a Random Forest Regression with Leave-One-Out Cross-Validation, which determines cell passage with good performance (MAE 1.094 and R2 0.897) and which is comparable to previous solutions. Using the advantage of BS-seq to analyze consecutive CpGs methylation patterns, we demonstrated that combining the analysis of average DNA methylation levels with local methylation heterogeneity scores—thereby reflecting stochastic DNA methylation dynamics—can improve the quality of the epigenetic clock models. Therefore, we propose a research strategy for creating customized epigenetic clocks using targeted BS-seq and provide a mechanistic conceptualization of how information on longitudinal changes in DNA methylation patterns can potentially be used for the assessment of specific aging aspects.

Bone is the most common site of prostate cancer metastasis, leading to significant morbidity, treatment resistance, and mortality. A major challenge in understanding treatment response is the complex, bone metastatic niche. Here, we report the first patient-specific microphysiological system (MPS) to incorporate six primary human stromal cell types found in the metastatic bone niche (mesenchymal stem cells, adipocytes, osteoblasts, osteoclasts, fibroblasts, and macrophages), alongside an endothelial microvessel, and prostate tumor epithelial spheroids in an optimized media that supports their viability and phenotype. We tested two standard of care drugs, darolutamide and docetaxel, in addition to sacituzumab govitecan (SG), currently in clinical trials for prostate cancer, demonstrating that the MPS accurately replicates androgen response sensitivity and captures stromal microenvironment-mediated resistance. This advanced MPS provides a robust platform for investigating the biological mechanisms of treatment response and for identification and testing of therapeutics to advance patient-specific MPS towards personalized clinical-decision making. The authors developed a patient-specific microphysiological system using the LumeNEXT microfluidic platform to more accurately recreate the prostate cancer metastatic bone tumor microenvironment.

Plastin-3 (PLS3) encodes T-plastin, an actin-bundling protein mediating the formation of actin filaments by which numerous cellular processes are regulated. Loss-of-function genetic defects in PLS3 are reported to cause X-linked osteoporosis and childhood-onset fractures. However, the molecular etiology of PLS3 remains elusive. Functional compensation by actin-bundling proteins ACTN1, ACTN4, and FSCN1 was investigated in zebrafish following morpholino-mediated pls3 knockdown. Primary dermal fibroblasts from six patients with a PLS3 variant were also used to examine expression of these proteins during osteogenic differentiation. In addition, Pls3 knockdown in the murine MLO-Y4 cell line was employed to provide insights in global gene expression. Our results showed that ACTN1 and ACTN4 can rescue the skeletal deformities in zebrafish after pls3 knockdown, but this was inadequate for FSCN1. Patients’ fibroblasts showed the same osteogenic transdifferentiation ability as healthy donors. RNA-seq results showed differential expression in Wnt1, Nos1ap, and Myh3 after Pls3 knockdown in MLO-Y4 cells, which were also associated with the Wnt and Th17 cell differentiation pathways. Moreover, WNT2 was significantly increased in patient osteoblast-like cells compared to healthy donors. Altogether, our findings in different bone cell types indicate that the mechanism of PLS3-related pathology extends beyond actin-bundling proteins, implicating broader pathways of bone metabolism.