Biology subjects

Hofmann, S.

Publications and source records attributed to Hofmann, S..

At least 19 recordsLinked to original sources

bioRxiv2026

Mechanoscribed Xolography for Multiscale Mechanical Patterning of Cell-laden Hydrogels

Mechanotransduction is a key regulator of healthy and malignant tissue development. However, current mechanobiological insights are largely derived from cells cultured on two dimensional substrates or in homogeneous biomaterials, which insufficiently capture the three dimensional (3D) heterogeneity and dynamic nature of the native extracellular matrix. This gap persists due to a lack of manufacturing processes and analysis modalities to control and monitor material properties and their evolution in 3D. Here, Mechanoscribed Xolography is introduced as a light sheet volumetric bioprinting process to rapidly fabricate 3D cell laden hydrogels with spatially controlled mechanical heterogeneity across multiple length scales. Moreover, synchronizing in-situ optical microscopy with printing enables autonomous detection and spatial location driven printing around cellular spheroids. The refinement and spatiotemporal evolution of material properties are assessed with Brillouin microscopy, which verifies that the contrast of imprinted heterogeneity is preserved over three weeks. Remarkably, regional differences in cell behavior are attained within a one-step, one-material construct with elasticity of approximately 0.3 kPa to 0.8 kPa, or 0.7 kPa to 2.9 kPa with a crosslinking-enhancing additive. This foundational work on high-precision, rapid bioprinting surrounding cellular objects represents a unique tool to systematically advance experimentation of 3D mechanotransduction and prospectively engineer cell instructive biomaterials for regenerative medicine.

bioengineering↗

bioRxiv2026

RNA splicing factor mutations drive myeloid neoplasm oncogenesis through protein complex poisoning

RNA splicing factor mutations (SFmut) are founding oncogenic events which cause RNA splicing errors with unpredictable gene expression dynamics. Despite extensive transcriptomic studies, SFmut cancer-initiating mechanisms remain elusive. Among SFmut cancers, myeloid neoplasms (MN) alone offer a setting where true cancer-driving SFmut stem cells can be identified, namely the hematopoietic stem cell (HSC). Within a cohort of 62 MN patients and 20 healthy donors, we conducted long/short-read single-cell transcriptomics (10X-ONT, n = 21) and immunophenotype-resolved low-cell proteomics (pauciproteomics, n = 78) to resolve gene expression, RNA splicing and protein expression dynamics across healthy and SFmut MN hematopoiesis. During hematopoietic differentiation, SFmut RNA mis-splicing decouples proteotranscriptomic dynamics in a mutation-specific manner. Pauciproteomics defines the functional effects of SFmut RNA mis-splicing on the protein layer, identifying early-stage protein effects which poison entire functional protein complexes and progressively disrupt the proteome-wide network during cellular maturation. Importantly, this information could neither be resolved nor predicted by transcriptomic analyses. Finally, we functionally validate the proteome dynamics of HSC through induced pluripotent stem cell culture models of early hematopoietic differentiation, identifying candidate mechanisms for SFmut oncogenesis. Overall, these data elucidate SFmut MN disease biology with unprecedented granularity, defining the molecular consequences of SFmut RNA mis-splicing and offering a generalizable framework for cancer stem cell studies.

cancer biology↗

bioRxiv2025

Comprehensive immune profiling of melanoma-draining lymph nodes identifies plasmacytoid dendritic cells as new biomarker for PD-L1 blockade

Immune checkpoint blockade (ICB) has become a powerful weapon in the treatment of melanoma and other cancer types. Still, only a minority of patients profits from this type of therapy. As ICB is associated with considerable adverse effects, predictive biomarkers to select patients for ICB treatment are urgently needed. Here, we comprehensively immunophenotyped primary tumors and tumor-draining lymph nodes (tdLNs) from a panel of ICB-resistant and -sensitive melanoma models in mice. Surprisingly, we found that whereas tumor-infiltrating CD8+ T cells showed strong responses to PD-L1 blockade in ICB-sensitive melanoma, CD8+ T cell profiles in tdLNs were very similar in ICB responder and non-responder models. In contrast, we identified distinct myeloid immune profiles in tdLNs correlating with ICB responsiveness, including a low frequency of activated dendritic cells and a high frequency of plasmacytoid dendritic cells (pDCs). Notably, pDC marker genes and frequencies in tdLNs of melanoma patients correlated with favorable outcome. Thus, the level of pDCs in tdLNs represents a potential new biomarker for ICB in melanoma patients.

immunology↗

bioRxiv2025

E2A SUMOylation promotes HAdV mediated inhibition of the tumor suppressor p53 during infection

HAdV express early viral genes to modulate the activity of the cellular tumor suppressor p53 and ensure efficient viral replication, through various processes, whilst some of them are not completely understood. HAdV oncoprotein E1B-55K interactions with SUMO, PML-IV/V and Sp100A are tightly connected to the transforming potential of the viral factor in non-lytic infections. SUMO modified HAdV DNA binding protein (DBP) E2A interacts with PML and Sp100A acting as a molecular bridge between viral replication centers (RCs) and PML tracks. Here, we provide a novel concept showing that HAdV E2A is involved in the inhibition of p53 activity and apoptosis; thus we identified a novel route of exhibiting virus induced oncogenic potential. We revealed an E1B-55K independent localization of p53 within HAdV replication centers marked by E2A. In the absence of any functional E1B-55K protein expression, p53 interacts with E2A. E2A SUMOylation promotes HAdV- mediated inhibition of p53/DNA binding and p53-dependent transactivation by supporting p53 degradation, E1B-55K interaction and colocalization with PML-V that leads to E1B-55K induced upregulation of p53 SUMOylation. In sum, we provide evidence for a novel mechanism by which the HAdV DBP E2A protein inhibits p53- mediated transcriptional activation and apoptotic host cell response. Intriguingly, these findings change the longstanding dogma of host DNA damage response (DDR) inactivation by HAdV infection and offer crucial insights to improve future p53 selective oncolytic virus therapies and adenoviral vectors.

cell biology↗

bioRxiv2024

Xolography for Biomedical Applications: Dual-color Light-sheet Printing of Hydrogels with Local Control over Shape and Stiffness

AbstractCurrent challenges in tissue engineering include creation of extracellular environments that support and interact with cells using biochemical, mechanical, and structural cues. Spatial control over these cues is currently limited due to a lack of suitable fabrication techniques. This study introduces Xolography, an emerging dual-color light-sheet volumetric printing technology, to achieve control over structural and mechanical features for hydrogel-based photoresins at micro-to macroscale while printing within minutes. We propose a water-soluble photoswitch photoinitiator system and are the first to demonstrate Xolography with a library of naturally-derived, synthetic, and thermoresponsive hydrogels. Centimeter-scale, three-dimensional constructs with positive features of 20 {micro}m and negative features of [~] 100 {micro}m are fabricated with control over mechanical properties (compressive moduli 0.2 kPa - 6.5 MPa). Notably, switching from binary to grayscaled light projection enables spatial control over stiffness (0.2 - 16 kPa). As a proof of concept, grayscaled Xolography is leveraged with thermoresponsive hydrogels to introduce reversible anisotropic shape changes beyond isometric shrinkage. We finally demonstrate Xolography of viable cell aggregates, laying the foundation for cell-laden printing of dynamic, cell-instructive environments with tunable structural and mechanical cues in a fast one-step process. Overall, these innovations unlock unique possibilities of Xolography across multiple biomedical applications.

bioengineering↗

bioRxiv2024

SF3B1-mutant mis-splicing of UBA1 confers a targetable therapeutic vulnerability through UBA1 inhibition

SF3B1 mutation-driven myelodysplastic syndromes (MDS-SF3B1) arise due to somatic mutation in the splicing factor SF3B1 gene. SF3B1 mutations induce RNA mis-splicing and loss of expression of critical genes for erythropoiesis, leading to erythroid dysplasia and ultimately refractory anemia. The development of precision medicine approaches for MDS- SF3B1 is hampered by the complexity of the mis-splicing landscape and its evaluation in disease-accurate model systems. To identify novel RNA mis-splicing events, isogenic SF3B1K700E and SF3B1WT iPSC lines from an MDS-SF3B1 patient were differentiated into hematopoietic cells in vitro and subjected to unsupervised splicing event analysis using full-length RNA sequencing data. This revealed SF3B1K700E-specific mis-splicing of ubiquitin-like modifier activating enzyme 1 (UBA1) transcripts, which encode the essential E1 protein at the apex of the ubiquitination cascade. UBA1 mis-splicing (UBA1ms) preserved UBA1ms mRNA but not protein expression. Consequently, UBA1ms diminished the pool of functional UBA1, sensitizing SF3B1K700E cell lines to the small-molecule UBA1 inhibitor TAK-243. Finally, analysis of CD34+ RNA sequencing data from an MDS patient cohort confirmed unique and ubiquitous UBA1ms in MDS-SF3B1 patients, without detection in other splicing factor-mutated MDS patients, or in healthy individuals. TAK-243 selectively targeted MDS-SF3B1 primary CD34+ cells and reduced mutant cell number in colony-forming unit studies. In contrast, normal hematopoietic progenitor cells were unaffected. Altogether, we here define UBA1ms as a novel therapeutic vulnerability in SF3B1-mutant cells, introducing UBA1 inhibition as a potential avenue for future MDS-SF3B1 treatments.

cancer biology↗

bioRxiv2024

Non-invasive optoacoustic imaging visualizes exercise-induced dermal revascularization in obese mice

Microcirculatory dysfunction in dermal (dWAT) and subcutaneous white adipose tissue (scWAT) of obese humans may predict cardio-metabolic disease progression. In-vivo visualization and monitoring of microvascular remodeling in these tissues remains challenging. We compared performance of multi-spectral optoacoustic tomography (MSOT) and raster-scanning optoacoustic mesoscopy (RSOM) in visualizing lipid and hemoglobin contrast in scWAT and dWAT of diet-induced obese (DIO) mice undergoing voluntary wheel running. MSOT quantitatively visualized lipid and hemoglobin contrast in fat depots at early stages of DIO. RSOM precisely visualizes microvasculature with quantitative readouts of skin layer thickness and vascular density in dWAT and dermis. Combination of MSOT and RSOM resolved exercise-induced morphological changes in microvasculature density, tissue oxygen saturation, lipid and blood volume content in dWAT and scWAT. Combination of MSOT and RSOM precisely monitor microcirculatory dysfunction and intervention response in dWAT and scWAT of DIO mice. Our findings lay out the foundation for future clinical studies using optoacoustic-derived vascular readouts from adipose tissues as a biomarker for monitoring microcirculatory function in cardio-metabolic disease.

bioengineering↗

bioRxiv2024

Graphene microelectrode arrays, 4D structured illumination microscopy, and a machine learning-based spike sorting algorithm permit the analysis of ultrastructural neuronal changes during neuronal signalling in a model of Niemann-Pick disease type C

Simultaneously recording network activity and ultrastructural changes of the synapse is essential for advancing our understanding of the basis of neuronal functions. However, the rapid millisecond-scale fluctuations in neuronal activity and the subtle sub-diffraction resolution changes of synaptic morphology pose significant challenges to this endeavour. Here, we use graphene microelectrode arrays (G-MEAs) to address these challenges, as they are compatible with high spatial resolution imaging across various scales as well as high temporal resolution electrophysiological recordings. Furthermore, alongside G-MEAs, we deploy an easy-to-implement machine learning-based algorithm to efficiently process the large datasets collected from MEA recordings. We demonstrate that the combined use of G-MEAs, machine learning (ML)-based spike analysis, and four-dimensional (4D) structured illumination microscopy (SIM) enables the monitoring of the impact of disease progression on hippocampal neurons which have been treated with an intracellular cholesterol transport inhibitor mimicking Niemann-Pick disease type C (NPC) and show that synaptic boutons, compared to untreated controls, significantly increase in size, which leads to a loss in neuronal signalling capacity.

neuroscience↗

bioRxiv2023

Culture system for longitudinal monitoring of bone remodeling ex vivo

To quantify and visualize both bone formation and resorption within osteochondral explants cultured ex vivo is challenging with the current analysis techniques. An approach that enables monitoring of bone remodeling dynamics is longitudinal micro-computed tomography ({micro}CT), a non-destructive technique that relies on repeated {micro}CT scanning and subsequent registration of consecutive scans. In this study, a two-compartment culture system suitable for osteochondral explants that allowed for {micro}CT scanning during ex vivo culture was established. Explants were scanned repeatedly in a fixed orientation, which allowed assessment of bone remodeling due to adequate image registration. Using this method, bone formation was found to be restricted to the outer surfaces when cultured statically. To demonstrate that the culture system could capture differences in bone remodeling, explants were cultured statically and under dynamic compression as loading promotes osteogenesis. No quantitative differences between static and dynamic culture were revealed. Still, only in dynamic conditions, bone formation was visualized on trabecular surfaces located within the inner cores, suggesting enhanced bone formation towards the center of the explants upon mechanical loading. Taken together, the ex vivo culture system in combination with longitudinal {micro}CT scanning and subsequent registration of images demonstrated potential for evaluating bone remodeling within explants.

bioengineering↗

bioRxiv2023

Integration of Osteoclastogenesis through addition of PBMCs in Human Osteochondral Explants cultured Ex vivo

The preservation of tissue specific cells in their native 3D extracellular matrix in bone explants provides a unique platform to study remodeling. Thus far, studies involving bone explant cultures showed a clear focus on achieving bone formation and neglected osteoclast activity and resorption. To simulate the homeostatic bone environment ex vivo, both key elements of bone remodeling need to be represented. This study aimed to assess and include osteoclastogenesis in human osteochondral explants through medium supplementation with RANKL and M-CSF and addition of peripheral blood mononuclear cells (PBMCs), providing osteoclast precursors. Osteochondral explants were freshly harvested from human femoral heads obtained from hip surgeries and cultured for 20 days in a two-compartment culture system. Osteochondral explants preserved viability and cellular abundance over the culture period, but histology demonstrated that resident osteoclasts were no longer present after 4 days of culture. Quantitative extracellular tartrate resistant acid phosphatase (TRAP) analysis confirmed depletion of osteoclast activity on day 4 even when stimulated with RANKL and M-CSF. Upon addition of PBMCs, a significant upregulation of TRAP activity was measured from day 10 onwards. Evaluation of bone loss trough {micro}CT registration and measurement of extracellular cathepsin K activity revealed indications of enhanced resorption upon addition of PBMCs. Based on the results we suggest that an external source of osteoclast precursors, such as PBMCs, needs to be added in long-term bone explant cultures to maintain osteoclastic activity, and bone remodeling. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/552563v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@b3df30org.highwire.dtl.DTLVardef@101d1e7org.highwire.dtl.DTLVardef@7bc56borg.highwire.dtl.DTLVardef@1de2d7e_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2023

Towards bone-remodeling-on-a-chip: self-assembling 3D osteoblast-osteoclast coculture in a microfluidic chip

Healthy bone is maintained by the process of bone remodeling. An unbalance in this process can lead to pathologies such as osteoporosis which are often studied with animal models. However, data from animals have limited power in predicting the results that will be obtained in human clinical trials. In search for alternatives to animal models, human in vitro models are emerging as they address the principle of reduction, refinement, and replacement of animal experiments (3Rs). At the moment, no complete in vitro model for bone-remodeling exists. Microfluidic chips offer great possibilities, particularly because of the dynamic culture options, which are crucial for in vitro bone formation. In this study, a scaffold free, fully human, 3D microfluidic coculture model of bone remodeling is presented. A bone-on-a-chip coculture system was developed in which human mesenchymal stromal cells differentiated into osteoblasts and self-assembled into scaffold free bone-like tissues with the shape and dimensions of human trabeculae. Human monocytes were able to attach to these tissues and to fuse into multinucleated osteoclast-like cells, establishing the coculture. Furthermore, a set-up was developed allowing for long-term (35 days) on-chip cell culture with benefits including continuous fluid-flow, low bubble formation risk, easy culture medium exchange inside the incubator and live cell imaging options. This on-chip coculture is a crucial advance towards developing in vitro bone remodeling models to facilitate drug testing.

bioengineering↗

bioRxiv2023

Towards development of serum substitute medium to induce osteoclast differentiation of human peripheral blood derived monocytes

Fetal bovine serum (FBS) is a widely used supplement in cell culture media despite its known drawbacks, including ethical, safety, and scientific issues. To overcome the drawbacks of using FBS in cell culture, a defined serum substitute medium needs to be developed. The development of such a medium depends on the cell type, which makes it impossible to use one universal serum substitute medium for all cells. Osteoclasts are large, multinucleated cells originated from the hematopoietic stem cell lineage that play an important role in regulating bone mass and quality. To date, no defined serum substitute medium formulations have been reported for osteoclast differentiation of monocytes derived from peripheral blood mononuclear cells (PBMCs). Here, we have attempted to develop such a serum substitute medium for the osteoclastogenesis process in a stepwise approach. Essential components were added to the medium while monocytes were cultured in 96-well plates and in Osteo-Assay well plates to analyze the formation of tartrate resistant acid phosphatase (TRAP) expressing multinucleated osteoclasts with distinct actin ring and to analyze the resorption activity of mature osteoclasts for 21 days, respectively. The serum substitute medium was aimed at supporting monocyte and later osteoclast survival, differentiation of monocytes towards multinucleated osteoclasts, and the resorption of mineralized matrix as a measure of functionality. All points were achieved after 21 days of culture in the developed serum substitute medium. This serum substitute medium could potentially replace FBS in osteoclastogenesis studies eliminating its debated use. Moreover, the well-defined serum substitute environment simplifies the study of factors released by the cells that were so far overwhelmed by the complexity of FBS.

bioengineering↗

bioRxiv2023

Matrix vesicle isolation from a three-dimensional in vitro bone model

Extracellular vesicles (EV) are nano-sized bilayer vesicles that are involved in biological functions and secreted by a wide variety of cells. Osteoblasts, the bone forming cells, can release a subset of EVs known as matrix vesicles (MtVs) which are believed to be involved in matrix mineralization and feature bone forming properties. Osteoblast-derived EVs or MtVs have been mostly isolated from conditions which are still far from nature, i.e. mesenchymal stromal cells (MSCs), or osteoblast cell lines cultured in two-dimensional (2D) tissue culture flasks. In our study, we aimed at investigating whether MtVs could also be isolated from an environment which better resembles the complex in vivo situation. This study investigated the EVs secretion during osteogenic differentiation of human bone marrow MSCs (hBMSCs) in the most advanced human three-dimensional (3D) in vitro woven bone constructs previously developed by our group. hBMSCs were cultured in spinner flask bioreactors which induced wall shear stress on cells and directed the cells to differentiate towards osteoblasts and osteocytes. The EVs secreted into the culture medium were isolated and characterized based on their morphological, biological, and functional properties. The characteristics of a part of isolated EVs shared similarities with MtVs. These vesicles were electron-dense and electron-lucent, showed alkaline phosphatase (ALP) activity, increased the amount of released free phosphate into the culture medium, and increased the amount of deposited phosphate within the ECM. The results indicate that a complex 3D environment mimicking bone development is favorable to stimulate MtV-producing cells to produce targeted MtVs in vitro. These MtVs potentially could be used as a biological agent for bone regeneration and fracture healing through, for instance, integration with biomaterials to target bone formation locally.

bioengineering↗

bioRxiv2022

The impact of culture variables on 3D human in vitro bone remodeling; a design of experiments approach

Human in vitro bone remodeling models, using osteoclast-osteoblast co-cultures, could facilitate the investigation of human healthy (i.e., balanced) and pathological (i.e., unbalanced) bone remodeling while reducing the need for animal experiments. Although current in vitro osteoclast-osteoblast co-cultures have improved our understanding of bone remodeling, they lack culture method and outcome measurement standardization, which is hampering reproducibility and translatability. Therefore, in vitro bone remodeling models could benefit from a thorough evaluation of the impact of culture variables on functional and translatable outcome measures, with the aim to reach healthy balanced osteoclast and osteoblast activity. Using a resolution III fractional factorial design, we identified the main effects of commonly used culture variables on bone turnover markers in a robust in vitro human bone remodeling model. Our model was able to capture physiological quantitative resorption - formation coupling along all conditions, whereby remodeling could be enhanced by external stimuli. Especially culture conditions of two runs showed promising results, where conditions of one run could be used as a high bone turnover system and conditions of another run as a self-regulating system as the addition of osteoclastic and osteogenic differentiation factors was not required for remodeling. The results generated with our in vitro model allow for better translation between in vitro studies and towards in vivo studies, for improved preclinical bone remodeling drug development. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=67 SRC="FIGDIR/small/516134v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@1068cc1org.highwire.dtl.DTLVardef@102d788org.highwire.dtl.DTLVardef@1942864org.highwire.dtl.DTLVardef@1e7d8b7_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2022

Development of serum substitute medium for bone tissue engineering

In tissue engineering, cells are grown often on scaffolds and subjected to chemical/mechanical stimuli. Most such cultures still use fetal bovine serum (FBS) despite its known disadvantages including ethical concerns, safety issues, and variability in composition, which greatly influences the experimental outcomes. To overcome the disadvantages of using FBS, chemically defined serum substitute medium needs to be developed. Development of such medium depends on cell type and application - which makes it impossible to define one universal serum substitute medium for all cells in any application. Here, we developed a serum substitute medium for bone tissue engineering (BTE) in a step-by-step process. Essential components were added to the medium while human bone marrow mesenchymal stromal cells (hBMSCs, osteoblast progenitor cells) were cultured in two-dimensional (2D) and three-dimensional (3D) substrates. In a 3-week culture, the developed serum substitute medium worked equally well as FBS containing medium in term of cell attachment to the substrate, cell survival, osteoblast differentiation, and deposition of extracellular matrix. In the next step, the use of serum substitute medium was evaluated when culturing cells under mechanical loading in the form of shear stress. The outcomes showed that the application of shear stress is essential to improve extracellular matrix formation while using serum substitute medium. The developed serum substitute medium could pave the way in replacing FBS for BTE studies eliminating the use of controversial FBS and providing a better-defined chemical environment for BTE studies. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=147 SRC="FIGDIR/small/511271v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@7bc741org.highwire.dtl.DTLVardef@5689fcorg.highwire.dtl.DTLVardef@17d1a3corg.highwire.dtl.DTLVardef@456844_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2022

A dialysis medium refreshment cell culture set-up for an osteoblast-osteoclast coculture

Culture medium exchange leads to loss of valuable auto- and paracrine factors produced by the cells. However, frequent renewal of culture medium is necessary for nutrient supply and to prevent waste product accumulation. Thus it remains the gold standard in cell culture applications. The use of dialysis as a medium refreshment method could provide a solution as low molecular weight molecules such as nutrients and waste products could easily be exchanged, while high molecular weight components such as growth factors, used in cell interactions, could be maintained in the cell culture compartment. This study investigates a dialysis culture approach for an in vitro bone remodeling model. In this model, both the differentiation of human mesenchymal stromal cells (MSCs) into osteoblasts and monocytes (MCs) into osteoclasts is studied. A custom-made simple dialysis culture system with a commercially available cellulose dialysis insert was developed. The data reported here revealed increased osteoblastic and osteoclastic activity in the dialysis groups compared to the standard non-dialysis groups, mainly shown by significantly higher alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) activity, respectively. This simple culture system has the potential to create a more efficient microenvironment allowing for cell interactions via secreted factors in mono- and cocultures and could be applied for many other tissues.

bioengineering↗

bioRxiv2022

Characterization of three-dimensional bone-like tissue growth and organization under influence of curvature and directional fluid flow

The transition in the field of bone tissue engineering from bone regeneration to three-dimensional in vitro models has come with the challenge of recreating a dense and anisotropic bone-like extracellular matrix with cell culture. The creation of such an organized bone-like extracellular matrix has received little attention thus far. Although the mechanism by which bone extracellular matrix gains its structure is not fully understood, curvature (especially concavities), mechanical loading due to deformations or directional fluid flow, and osteocyte signaling have been identified as potential contributors. Here, guided by computational simulations, we evaluated three-dimensional cell and bone-like tissue growth and organization in a concave channel with and without directional fluid flow stimulation. Human bone-marrow derived mesenchymal stromal cells were seeded on donut-shaped silk fibroin scaffolds and stimulated to undergo osteogenic differentiation for 42 days statically or in a flow perfusion bioreactor. Constructs were investigated for cell distribution, and tissue growth and organization on day 14, 28, and 42. As a result, directional fluid flow was able to improve bone-like tissue growth but not organization. After 28 days of culture, when osteogenic differentiation was likely accomplished, cells tended to have a small preference for orientation in the tangential (i.e., circumferential) direction of the channel. Based on our results, we suggest that three-dimensional bone-like tissue anisotropy might be guided by curvature, while extracellular matrix production can be increased through the application of fluid shear stress. With this study, an initial attempt in three-dimensions was made to improve the resemblance of in vitro produced bone-like extracellular matrix to the physiological bone extracellular matrix.

bioengineering↗

bioRxiv2022

Fasting-sensitive SUMO-switch on Prox1 controls hepatic cholesterol metabolism

The liver is the major metabolic hub, ensuring appropriate nutrient supply during fasting and feeding. In obesity, accumulation of excess nutrients hampers proper liver function and is linked to non-alcoholic fatty liver disease. Understanding the signaling mechanisms that enable hepatocytes to quickly adapt to dietary cues, might help to restore balance in liver diseases. Post-translational modification by attachment of the Small Ubiquitin-like Modifier (SUMO), allows for a dynamic regulation of numerous processes including transcriptional reprograming. Here, we demonstrate that the specific SUMOylation of transcription factor Prox1 represents a nutrient-sensitive determinant of hepatic fasting metabolism. Prox1 was highly modified by SUMOylation on lysine 556 in the liver of ad libitum and re-fed mice, while this modification was strongly abolished upon fasting. In a context of diet-induced obesity, Prox1 SUMOylation became insensitive to fasting cues. Hepatocyte-selective knock in of a SUMOylation-deficient Prox1 mutant into mice fed a high fat/high fructose diet led to reduction of systemic cholesterol levels, associated with the induction of bile acid detoxifying pathways in mutant livers during fasting. As appropriate and controlled fasting protocols have been shown to exert beneficial effects on human health, tools to maintain the nutrient-sensitive SUMOylation switch on Prox1 may thus contribute to the development of "fasting-based" approaches for the maintenance of metabolic health.

physiology↗