Biology subjects

Diekman, B. O.

Publications and source records attributed to Diekman, B. O..

13 recordsLinked to original sources

bioRxiv2026

Mapping the response of primary human chondrocytes to an osteoarthritis-relevant stimulus by single-cell RNA sequencing

Objective: To determine how fibronectin fragment (Fn-f), a matrikine that induces an osteoarthritis (OA)-like phenotype, reshapes chondrocyte states and transcriptional programs at single-cell resolution. Design: Primary human ankle chondrocytes from two cadaveric donors without OA were treated with PBS or purified recombinant Fn-f for 18 hours and profiled by single-cell RNA sequencing. Transcriptional states were characterized by marker genes and functional enrichment, compared with classifications used for OA chondrocytes, and evaluated for Fn-f-dependent changes in abundance and transcriptional programs. Results: Five transcriptionally distinct populations were identified with features that matched previous classifications of effector, inflammatory, pre-hypertrophic/fibrochondrocyte, reparative, and homeostatic chondrocytes. Fn-f markedly redistributed cells among these states, enriching inflammatory chondrocytes while reducing effector and reparative populations. Within transcriptional states, Fn-f differentially altered matrix/cartilage, inflammatory, and stress/immediate-early programs. These responses did not uniformly correspond to assigned subtype identities, demonstrating that transcriptional programs can be dynamically deployed across chondrocyte states. Conclusions: Controlled perturbations of healthy chondrocytes complement observational OA atlases by resolving stimulus-dependent changes in chondrocyte state abundance and transcriptional programs. Discordance between subtype annotations and dynamic functional programs further highlights the need for harmonized definitions of chondrocytes across phenotypic states.

genetics↗

bioRxiv2026

Human hip osteoarthritis-associated Chadl variant induces intervertebral disc degeneration in mice

Chondroadherin-like (CHADL), a small leucine-rich proteoglycan, plays a role in ECM assembly and chondrocyte differentiation. CHADL has been implicated in osteoarthritis (OA) due to the discovery of a rare frameshift variant (rs532464664) that confers one of the highest genetic risks for hip OA reported to date. In this study, we characterized the skeletal phenotypes of two mouse models of Chadl loss-of-function, a global null and an 8bp insertion that mimics the human frameshift variant. Surprisingly, while there was a slight increase in knee OA at 12 months, there were no genotype-based differences in knee or hip OA in aged mice. Interestingly, histological assessment revealed phenotypic changes in the intervertebral discs of mutant mice, along with increased disc height index, suggesting altered motion-segment biomechanics. Polarized imaging showed dysregulated collagen turnover in the annulus fibrosus (AF), evidenced by changes in the proportions of thin and intermediate fibers. There was decreased abundance of the nucleus pulposus (NP) marker CA3 and decreased staining for ECM proteins in the AF of mutants. CHADL loss affected vertebral trabecular architecture, cortical thickness, and mineral density. This study highlights an unrecognized role of CHADL in fine-tuning ECM homeostasis and health of the intervertebral disc and vertebral bone.

cell biology↗

bioRxiv2026

CRISPR-mediated engineering of bovine satellite cells for Alpha-Gal Syndrome-compatible cultivated meat

Alpha-gal Syndrome (AGS) is a potentially life-threatening allergy caused by an IgE-mediated immune response to galactose--1,3-galactose (alpha-gal), a carbohydrate epitope present in most mammalian meats. Currently, strict avoidance of mammalian meat remains the primary management strategy for affected individuals, and alpha-gal-free beef is not commercially available. Here, we leverage cultivated meat as a biotechnology plat-form to address this unmet clinical need by engineering alpha-gal-free bovine muscle cells. Using CRISPR/Cas9 genome editing, we disrupted GGTA1, the gene encoding 1,3-galactosyltransferase, in immortalized bovine satellite cells (iBSCs). High-efficiency editing produced clonal GGTA1 knockout iBSCs harboring a homozygous frameshift mutation. Flow cytometry and immunofluorescence confirmed loss of the alpha-gal epitope, while bulk RNA-seq indicated minimal disruption of global gene expression and preserved myogenic differentiation capacity. Importantly, lysates from GGTA1 knockout iBSCs elicited substantially reduced basophil activation in assays using plasma from a patient with AGS, indicating reduced basophil activation consistent with reduced allergenic potential. Together, these findings establish a proof of concept for engineering AGS-compatible cultivated meat and demonstrate the potential of cultivated meat technologies to address human health challenges. HIGHLIGHTS{circ} CRISPR/Cas9-mediated disruption of GGTA1 eliminated alpha-gal from bovine satellite cells {circ}GGTA1 knockout cells retained myogenic identity and differentiation capacity {circ}GGTA1 knockout reduced basophil activation in an alpha-gal syndrome immune assay {circ}Genome-edited bovine cells provide a proof of concept for AGS-compatible cultivated meat

bioengineering↗

bioRxiv2026

Comparative whole-genome analyses of articular chondrocytes and skin fibroblasts reveal distinct genome instability landscapes in mesenchymal cell types

DNA damage lesions can result in mutations and genome rearrangements that are associated with cellular aging and diseases. The landscape of somatic mutations in individual tissue and cell types are dictated by their unique physiological states, cellular functions, mutagenic exposures, and efficiency of DNA repair. Articular chondrocytes and skin fibroblasts are two cell types of mesodermal origin with distinct exposure to internal and external sources of DNA damage. While somatic genome instability features of skin fibroblasts have been well detailed, knowledge about mechanisms underlying genome changes in chondrocytes is scarce. Here, we took a whole-genome sequencing approach to evaluate the load, sources, and patterns of genome changes in 18 primary human chondrocyte clones from donors with and without osteoarthritis (OA). Findings in chondrocyte clones largely agreed with a recent study of 100 single-cell sequenced chondrocytes. We compared genome changes in chondrocytes with clonally-expanded human skin fibroblasts sequenced in our previous studies. We demonstrated that skin fibroblasts show a higher burden of somatic mutations, with an increased rate of mutation accumulation per cell division. Motif-centered analyses of mutation catalogues identified only endogenous sources of mutations in chondrocytes, as opposed to skin fibroblasts which also showed a heavy burden of UV-induced mutations. Spontaneous deamination of meCpG and mutagenesis by exposure to small epoxides and SN2 electrophiles showed higher mutagenic activities in chondrocytes compared to skin fibroblasts. Chondrocytes showed ubiquitous prevalence of indels in homonucleotide runs of [≥]5 bases, while skin fibroblasts showed high contributions of UV-associated deletions of [≥]5bp not in repeats. Structural variants in rearrangement hotspots colocalized with human common fragile sites in skin fibroblasts, but not in chondrocytes. Together, our study comprehensively recorded genome instability features in chondrocytes and highlighted the unique mutagenesis landscapes of two mesenchymal cell types.

genetics↗

bioRxiv2025

ZNF865 (BLST) Regulates Human Cell Senescence and DNA Damage

Senescence has been shown to contribute to the progression of aging related diseases including degenerative disc disease (DDD). However, the mechanisms regulating senescence in the intervertebral disc (IVD) and other tissues/diseases remain poorly understood. Recently, ZNF865 (BLST) was identified as a previously uncharacterized zinc finger protein, that regulates a wide array of genes related to protein processing, cell senescence and DNA damage repair. Here, we show that ZNF865 expression decreases with age and pathology in human and mouse IVD samples. Utilizing CRISPR-guided gene modulation, we show that ZNF865 is necessary for healthy cell function and is a critical protein in regulating senescence and DNA damage in IVD cells, with implications for a wide range of tissues and organs. We also demonstrate that downregulation of ZNF865 induces senescence in asymptomatic human nucleus pulposus (NP) cells. Conversely, restoration in degenerative NP cells mitigates senescence, SASP expression and DNA damage, enhances ECM anabolism, and restores chromatin accessibility and gene expression to a healthy state. In vivo, CRISPRi of ZNF865 induces disc degeneration and painful behaviors. Collectively, our findings establish ZNF865 as a novel regulator of genome stability and a potential therapeutic target for mediating senescence/DNA damage in aging related diseases.

bioengineering↗

bioRxiv2024

Response splicing QTLs in primary human chondrocytes identifies putative osteoarthritis risk genes

Osteoarthritis affects millions worldwide, yet effective treatments remain elusive due to poorly understood molecular mechanisms. While genome-wide association studies (GWAS) have identified over 100 OA-associated loci, identifying the genes impacted at each locus remains challenging. Several studies have mapped expression quantitative trait loci (eQTL) in chondrocytes and colocalized them with OA GWAS variants to identify putative OA risk genes; however, the degree to which genetic variants influence OA risk via alternative splicing has not been explored. We investigated the role of alternative splicing in OA pathogenesis using RNA-seq data from 101 human chondrocyte samples treated with PBS (control) or fibronectin fragment (FN-f), an OA trigger. We identified 590 differentially spliced genes between conditions, with FN-f inducing splicing events similar to those in primary OA tissue. We used CRISPR/Cas9 to mimic an SNRNP70 splicing event observed in OA and FN-f-treated chondrocytes and found that it induced an OA-like expression pattern. Integration with genotyping data revealed 7,188 splicing quantitative trait loci (sQTL) affecting 3,056 genes. While many sQTLs were shared, we identified 738 and 343 condition-specific sQTLs for control and FN-f, respectively. We identified 15 RNA binding proteins whose binding sites were enriched at sQTL splice junctions and found that expression of those RNA binding proteins correlated with exon inclusion. Colocalization with OA GWAS identified 6 putative risk genes, including a novel candidate, PBRM1. Our study highlights the significant impact of alternative splicing in OA and provides potential therapeutic targets for future research.

genetics↗

bioRxiv2024

Multiplexed single-cell imaging reveals diverging subpopulations with distinct senescence phenotypes during long-term senescence induction.

Cellular senescence is a phenotypic state that contributes to the progression of age-related disease through secretion of pro-inflammatory factors known as the senescence associated secretory phenotype (SASP). Understanding the process by which healthy cells become senescent and develop SASP factors is critical for improving the identification of senescent cells and, ultimately, understanding tissue dysfunction. Here, we reveal how the duration of cellular stress modulates the SASP in distinct subpopulations of senescent cells. We used multiplex, single-cell imaging to build a proteomic map of senescence induction in human epithelial cells induced to senescence over the course of 31 days. We map how the expression of SASP proteins increases alongside other known senescence markers such as p53, p21, and p16INK4a. The aggregated population of cells responded to etoposide with an accumulation of stress response factors over the first 11 days, followed by a plateau in most proteins. At the single-cell level, however, we identified two distinct senescence cell populations, one defined primarily by larger nuclear area and the second by higher protein concentrations. Trajectory inference suggested that cells took one of two discrete molecular paths from unperturbed healthy cells, through a common transitional subpopulation, and ending at the discrete terminal senescence phenotypes. Our results underscore the importance of using single-cell proteomics to identify the mechanistic pathways governing the transition from senescence induction to a mature state of senescence characterized by the SASP.

cell biology↗

bioRxiv2024

Response eQTLs, chromatin accessibility, and 3D chromatin structure in chondrocytes provide mechanistic insight into osteoarthritis risk

Osteoarthritis (OA) poses a significant healthcare burden with limited treatment options. While genome-wide association studies (GWAS) have identified over 100 OA-associated loci, translating these findings into therapeutic targets remains challenging. Integrating expression quantitative trait loci (eQTL), 3D chromatin structure, and other genomic approaches with OA GWAS data offers a promising approach to elucidate disease mechanisms; however, comprehensive eQTL maps in OA-relevant tissues and conditions remain scarce. We mapped gene expression, chromatin accessibility, and 3D chromatin structure in primary human articular chondrocytes in both resting and OA-mimicking conditions. We identified thousands of differentially expressed genes, including those associated with differences in sex and age. RNA-seq in chondrocytes from 101 donors across two conditions uncovered 3782 unique eGenes, including 420 that exhibited strong and significant condition-specific effects. Colocalization with OA GWAS signals revealed 13 putative OA risk genes, 10 of which have not been previously identified. Chromatin accessibility and 3D chromatin structure provided insights into the mechanisms and conditional specificity of these variants. Our findings shed light on OA pathogenesis and highlight potential targets for therapeutic development. Highlights{circ} Comprehensive analysis of sex- and age-related global gene expression in human chondrocytes revealed differences that correlate with osteoarthritis {circ}First response eQTLs in chondrocytes treated with an OA-related stimulus {circ}Deeply sequenced Hi-C in resting and activated chondrocytes helps connect OA risk variants to their putative causal genes {circ}Colocalization analysis reveals 13 (including 10 novel) putative OA risk genes

genetics↗

bioRxiv2023

SIRT6 activation rescues the age-related decline in DNA damage repair in primary human chondrocytes

While advanced age has long been recognized as the greatest risk factor for osteoarthritis (OA), the biological mechanisms behind this connection remain unclear. Previous work has demonstrated that chondrocytes from older cadaveric donors have elevated levels of DNA damage as compared to chondrocytes from younger donors. The purpose of this study was to determine whether a decline in DNA repair efficiency is one explanation for the accumulation of DNA damage with age, and to quantify the improvement in repair with activation of Sirtuin 6 (SIRT6). Using an acute irradiation model to bring the baseline level of all donors to the same starting point, this study demonstrates a decline in repair efficiency during aging when comparing chondrocytes from young ([≤]45 years old), middle-aged (50-65 years old), or older (>70 years old) cadaveric donors with no known history of OA or macroscopic cartilage degradation at isolation. Activation of SIRT6 in middle-aged chondrocytes with MDL-800 (20 M) improved the repair efficiency, while inhibition with EX-527 (10 M) inhibited the rate of repair and the increased the percentage of cells that retained high levels of damage. Treating chondrocytes from older donors with MDL-800 for 48 hours significantly reduced the amount of DNA damage, despite this damage having accumulated over decades. Lastly, chondrocytes isolated from the proximal femurs of mice between 4 months and 22 months of age revealed both an increase in DNA damage with aging, and a decrease in DNA damage following MDL-800 treatment.

cell biology↗

bioRxiv2022

Patient-derived extracellular matrix demonstrates role of COL3A1 in blood vessel mechanics

Vascular Ehlers-Danlos Syndrome (vEDS) is a rare autosomal dominant disease caused by mutations in the COL3A1 gene, which renders patients susceptible to aneurysm and arterial dissection and rupture. To determine the role of COL3A1 variants in the biochemical and biophysical properties of human arterial ECM, we developed a method for synthesizing ECM directly from vEDS donor fibroblasts. We found that the protein content of the ECM generated from vEDS donor fibroblasts differed significantly from ECM from healthy donors, including upregulation of collagen subtypes and other proteins related to ECM structural integrity. We further found that ECM generated from a donor with a glycine substitution mutation was characterized by increased glycosaminoglycan content and unique viscoelastic mechanical properties, including increased time constant for stress relaxation, resulting in a decrease in migratory speed of human aortic endothelial cells when seeded on the ECM. Collectively, these results demonstrate that causal COL3A1 mutations lead to the synthesis of ECM that differs in composition, structure, and mechanical properties from healthy donors. These results further suggest that ECM mechanical properties could serve as a prognostic indicator for patients with vEDS, and the insights provided by the approach demonstrate the broader utility of cell-derived ECM in disease modeling.

bioengineering↗

bioRxiv2022

3D Chromatin Structure in Chondrocytes Identifies Putative Osteoarthritis Risk Genes

Genome-wide association studies (GWAS) have identified over 100 loci associated with osteoarthrtis (OA) risk, but the majority of OA risk variants are non-coding, making it difficult to identify the impacted genes for further study and therapeutic development. To address this need, we used a multi-omic approach and genome editing to identify and functionally characterize potential OA risk genes. Computational analysis of GWAS and ChIP-seq data revealed that chondrocyte regulatory loci are enriched for OA risk variants. We constructed a chondrocyte specific regulatory network by mapping 3D chromatin structure and active enhancers in human chondrocytes. We then intersected these data with our previously collected RNA-seq dataset of chondrocytes responding to fibronectin fragment (FN-f), a known OA trigger. Integration of the three genomic datasets with recently reported OA GWAS variants revealed a refined set of putative causal OA variants and their potential target genes. One of the novel putative target genes identified was SOCS2, which was connected to a putative causal variant by a 170 Kb loop and is differentially regulated in response to FN-f. CRISPR-Cas9-mediated deletion of SOCS2 in primary human chondrocytes from three independent donors led to heightened expression of inflammatory markers after FN-f treatment. These data suggest that SOCS2 plays a role in resolving inflammation in response to cartilage matrix damage and provides a possible mechanistic explanation for its influence on OA risk. In total, we identified 56 unique putative OA risk genes for further research and potential therapeutic development.

genomics↗

bioRxiv2020

The combination of mitogenic stimulation and cell cycle arrest induces senescence in equine and human cartilage explants

ObjectiveCellular senescence is a phenotypic state characterized by stable cell-cycle arrest, enhanced lysosomal activity, and the secretion of inflammatory molecules and matrix degrading enzymes. Senescence has been implicated in osteoarthritis (OA) pathophysiology; however, the mechanisms that drive senescence induction in cartilage and other joint tissues are unknown. While numerous physiological signals are capable of initiating the senescence phenotype, one emerging theme is that growth-arrested cells convert to senescence in response to sustained mitogenic stimulation. The goal of this study was to develop an in vitro articular cartilage explant model to investigate the mechanisms of senescence induction. DesignThis study utilized healthy articular cartilage derived from cadaveric equine stifles and human ankles. Explants were irradiated or treated with palbociclib to initiate cell cycle arrest, and mitogenic stimulation was provided by serum-containing medium (horse) and the inclusion of growth factors (human). The primary readout of senescence was a quantitative flow cytometry assay to detect senescence-associated {beta} galactosidase activity (SA-{beta}-gal). ResultsIrradiation of equine explants caused 25.39% of cells to express high levels of SA-{beta}-gal, as compared to 3.82% in control explants (p=0.0031). For human cartilage, explants that received both mitogenic stimulation and cell cycle arrest showed increased rates of senescence induction as compared to baseline control (7.16% vs. 2.34% SA-{beta}-gal high, p=0.0007). ConclusionsTreatment of cartilage explants with mitogenic stimuli in the context of cell-cycle arrest reliably induces high levels of SA-{beta} gal activity, which provides a physiologically relevant model system to investigate the mechanisms of senescence induction.

cell biology↗

bioRxiv2019

Engineered cartilage from human chondrocytes with homozygous knockout of cell cycle inhibitor p21

Risk factors for the development of osteoarthritis (OA) include genetic background and focal cartilage injury. The search for disease-modifying OA therapies would benefit from a more comprehensive knowledge of the genetic variants that contribute to chondrocyte dysfunction and the barriers to cartilage regeneration. One goal of this study was to establish a system for producing engineered cartilage tissue from genetically-defined primary human chondrocytes through genome editing and single-cell expansion. This process was utilized to investigate the functional effect of bi-allelic knockout of the cell cycle inhibitor p21. The use of ribonucleoprotein (RNP) CRISPR/Cas9 complexes targeting two sites in the coding region of p21 resulted in a high frequency (16%) of colonies with homozygous p21 knockout. Chondrogenic pellet cultures from expanded chondrocytes with complete loss of p21 produced more glycosaminoglycans (GAG) and maintained a higher cell number. Single-cell derived colonies retained the potential for robust matrix production after expansion, allowing for analysis of colony variability from the same population of targeted cells. The effect of enhanced cartilage matrix production in p21 knockout chondrocytes persisted when matrix production from individual colonies was analyzed. Chondrocytes had lower levels of p21 protein with further expansion, and the difference in GAG production with p21 knockout was strongest at early passages. These results support previous findings that implicate p21 as a barrier to cartilage matrix production and regenerative capacity. Further, this work establishes the use of genome-edited human chondrocytes as a promising approach for engineered tissue models containing user-defined gene knockouts and other genetic variants for investigation of OA pathogenesis.

bioengineering↗