Biology subjects

Kilian, K. A.

Publications and source records attributed to Kilian, K. A..

8 recordsLinked to original sources

bioRxiv2025

Gene-Corrected Basal Cells Restore CFTR In Vitro; Transplants Regenerate Epithelium in a Preclinical Sinus Model

BackgroundCystic fibrosis (CF) is caused by mutations in the CFTR gene, leading to epithelial dysfunction and progressive lung disease. Although CFTR modulators have transformed care, [~]10% of people with CF remain without effective therapy. Durable, mutation-agnostic approaches are urgently needed. MethodWe used a lentiviral (LV) vector to deliver wild-type CFTR to airway basal cells derived from 13 paediatric CF participants with a range of genotypes. Transduced cells were assessed for transgene expression, epithelial differentiation, and CFTR function using air-liquid interface (ALI) cultures. Separately, to evaluate regenerative capacity in vivo, LVGFP-transduced rabbit airway basal cells were transplanted into the denuded nasal septum of healthy New Zealand white rabbits using a biocompatible scaffold. ResultsTransduced basal cells retained multilineage differentiation capacity, forming well-organized, pseudostratified epithelium with intact barrier function and ciliary activity. CFTR channel activity was restored to levels comparable to or exceeding those achieved with elexacaftor/tezacaftor/ivacaftor (ETI), including in individuals with nonsense mutations. Combined CFTR transduction plus ETI treatment showed additive benefit. In vivo, transplanted rabbit basal cells engrafted and differentiated to regenerate a mucociliary epithelium, with improved nasal potential difference and mucociliary clearance compared to scaffold-only controls. ConclusionOur study demonstrates that LV-mediated CFTR gene addition restores CFTR function in vitro across genotypes and supports epithelial regeneration in a clinically relevant animal airway model. This two-part platform offers a scalable path toward cell therapies for all people with CF and may have broader applications in upper airway epithelial repair.

cell biology↗

bioRxiv2024

Tuning mechanical milieux of tissue templates and their cellular inhabitants to guide mechanoadaptation

Mechanomics describes the adaptation of mesenchymal stem cells (MSCs) to their mechanical environment, via cytoskeletal remodeling, as well as changes in shape and volume, ultimately resulting in emergent lineage commitment. Here we elucidated effects of exogenous microtubule stabilization, using paclitaxel (PAX), on stem cells capacity to sense and adapt to changes in their local mechanical environment. We studied the interplay between the living, evolving cells and their mechanical environment using established experimental and computational tools for respective delivery and prediction of shape and volume changing stresses. Stiffened and volumetrically larger microtubule-stabilized MSCs and their experienced significantly different normal and shear stress compared to control cells when exposed to identical bulk laminar flow (0.2 dyn/cm2) for one hour. These spatiotemporal mechanical cues transduced to the nucleus via the cytoskeleton, triggering significantly different changes in gene expression indicative of emergent lineage commitment than those observed in control cells. Using a paired computational model, we further predicted a range of mechanoadaptation responses of microtubule-stabilized cells to scaled up flow magnitudes (1 and 2 dyn/cm2). Hence, MSCs adapt to as well as modulate their own mechanical environment via cytoskeletal remodeling and lineage commitment - microtubule stabilization changes not only MSCs mechanoadaptive machinery, their capacity to adapt, and their lineage commitment, but also their mechanical environment. Taken as a whole, these studies corroborate our working hypothesis that MSCs and their mechanoadaptive machinery serve as sensors and actuators, intrinsically linked to their lineage potential via mechanoadaptive feedback loops which are sensitive to exogenous modulation via biochemical and biophysical means. ClassificationBiological Systems Engineering, Computational Simulations, Cell Biology, Biophysics

bioengineering↗

bioRxiv2024

Stem cell mechanoadaptation - Part A - Effect of microtubule stabilization and volume changing stresses on cytoskeletal remodeling

Here we report on the first part of a two-part experimental series to elucidate spatiotemporal cytoskeletal remodeling, which underpins the evolution of stem cell shape and fate, and the emergence of tissue structure and function. In Part A of these studies, we first develop protocols to stabilize microtubules exogenously using paclitaxel (PAX) in a standardized model murine embryonic stem cell line (C3H/10T1/2) to maximize comparability with previous published studies. We then probe native and microtubule stabilized stem cells capacity to adapt to volume changing stresses effected by seeding at increasing cell densities, which emulates local compression and tissue template formation during development. Within the concentration range 1 - 100 nM, microtubule stabilized stem cells maintain viability and reduce proliferation. PAX-stabilization of microtubules is associated with increased cell volume as well as flattening of the cell and nucleus. Compared to control cells, microtubule stabilized cells exhibit thick, bundled microtubules and highly aligned, thicker and longer F-actin fibers, corresponding to an increase in the Youngs Modulus of the cell. Both F-actin and microtubule concentration increase with increasing PAX concentration, whereby the increase in F-actin is more prominent in the basal region of the cell. The corresponding increase in microtubule is observed more globally across the apical and basal region of the cell. Seeding at increasing target densities induces local compression on cells. This increase in local compression modulates cell volume and concomitant increases in F-actin and microtubule concentration to a greater degree than microtubule stabilization via PAX. Cells seeded at high density (HD) exhibit higher bulk modulus than corresponding cells seeded at low density (LD). These data demonstrate the capacity of stem cells to adapt to an interplay of mechanical and chemical cues, i.e. respective compression and exogenous microtubule stabilization; the resulting cytoskeletal remodeling manifests as evolution of mechanical properties relevant to development of multicellular tissue constructs. Significance statementElucidation of mechanisms by which stem cells adapt across length and time scales may prove enabling for the development of regenerative medicine therapies and devices that emulate natural processes. Dynamic cytoskeletal remodeling underpins the emergence of structure-function relationships at the tissue length scale. Here we stabilized the tubulin cytoskeleton exogenously using paclitaxel (PAX), a microtubule depolymerization inhibitor. We probed stem cell mechanoadaptation by seeding at increasing density to introduce local compression to cells. Changes in cytoskeletal architecture and concentration of F-actin and tubulin per cell occurred in a PAX concentration-dependent manner. Compression from increasing seeding density modulated this PAX-induced cytoskeletal remodeling and mechanical properties of the multicellular constructs. Hence, mechanical cues counterbalance concentration-dependent effects of exogenous chemical microtubule stabilization.

cell biology↗

bioRxiv2024

Stem cell mechanoadaptation Part B - Microtubule stabilization and substrate compliance effects on cytoskeletal remodeling

Stem cells adapt to their local mechanical environment by rearranging their cytoskeleton, which underpins the evolution of their shape and fate, as well as the emergence of tissue structure and function. Here we report on the second part of a two-part experimental series to elucidate spatiotemporal cytoskeletal remodeling and resulting changes in morphology and mechanical properties of cells, their nuclei, akin to mechanical testing of the most basic living and adapting unit of life, in situ in model tissue templates. We probed the native and PAX-exposed (inhibiting cytoskeleton tubulin depolymerization) stem cells cytoskeletal adaptation capacity on substrates of different compliance (exerting local tension on cells) and in combination with exposure to local compression effected with increased target seeding densities (5000 cells/cm2 - Low Density, LD; 15,000 cells/cm2, High Density, HD). On 10 and 100 kPa gels, cells seeded at both LD and cells proliferated to HD exhibited bulk moduli that nearly matched those of their respective substrates, hence exhibiting a greater increase in Youngs Modulus after microtubule stabilization than cells cultured on glass. Culture on compliant substrates also reduced the PAX-mediated F-actin and microtubule concentration increase. On gels, F-actin alignment decreased as more randomly oriented, short actin crosslinks were observed, representing emergent adaptation to the compliant substrate, mediated through myosin II contractility. We conclude that stem cell adaptation to compliant substrates facilitates the accommodation of larger loads from the PAX-stabilized polymerizing microtubule, which in turn exerts a larger effect in determining cells capacity to stiffen and remodel the cytoskeleton. Taken as a whole, these studies establish correlations between cytoskeleton and physical and mechanical parameters of stem cells that progress our understanding of the dynamic cytoskeleton, as well as shape changes in cells and their nuclei, culminating in emergent tissue development and healing. Significance StatementStem cells adapt to their dynamic environment by means of cytoskeleton rearrangements - underpinning the emergence of tissue structure-function relationship; this represents a current gap in knowledge that needs to be addressed, to better target tissue neogenesis and healing in context of regenerative medicine. We introduced compression via increasing seeding density and tension via compliant substrates to create tissue templates, while stabilizing microtubules. We found that mechanical and biophysical cues exert a greater effect in modulating cytoskeletal adaptation than exogenous chemical agents targeting the cytoskeleton, thus counterbalancing the concentration-dependent effect on cell physical and mechanical properties. We further found that stem cells with stabilized microtubules are sensitive to a range of substrate stiffness and seeding density that allowed cells or multicellular constructs to broaden their capacity to adapt their mechanical properties.

cell biology↗

bioRxiv2024

Enzyme Bioink for the 3D Printing of Biocatalytic Materials

The field of 3D biofabrication faces major challenges on the road to printing fully functional tissues and organs. One of them is adding functionality to the newly formed tissue for replicating an active biochemical environment. Native extracellular matrices sequester numerous bioactive species, making the microenvironment biochemically active. On the other hand, most 3D-printed constructs have limited activity, serving merely as mechanical scaffolding. Here we demonstrate active scaffolding through the integration of biocatalytic enzymes within the bioink. Enzymes are an attractive class of biocompatible and substrate-specific bioactive agents that can improve tissue regeneration outcomes. However, the difficulty in the application remains in providing enzymes at the targeted site in adequate amounts over an extended time. In this work, a durable biocatalytic active enzyme bioink for 3D extrusion-based bioprinting is developed by covalently attaching the globular enzyme horseradish peroxidase (HRP) to a gelatin methacrylate (Gel-MA) biopolymer scaffold. Upon introducing methacrylate groups on the surface of the enzyme, it undergoes photo-crosslinking in a post-printing step with the methacrylate groups of Gel-MA without compromising its activity. As a result, HRP becomes a fixed part of the hydrogel network and achieves higher stability inside the gel which results in a higher concentration and catalytic activity for a longer time than solely entrapping the protein inside the hydrogel. We also demonstrate the cytocompatibility of this enzyme bioink and show its printing capabilities for precise applications in the field of tissue engineering. Our approach offers a promising solution to enhance the bioactive properties of 3D-printed constructs, representing a critical step towards achieving functional biofabricated tissues.

bioengineering↗

bioRxiv2022

Gas modulating microcapsules for spatiotemporal control of hypoxia

Oxygen is a vital molecule involved in regulating development, homeostasis, and disease. The oxygen levels in tissue vary from 1 to 8% with deviations having major biological consequences. In this work, we developed an approach to encapsulate enzymes and nanozymes, at an unprecedented loading capacity, which precisely controls the oxygen content in cell culture. Here, a single microcapsule is able to locally perturb the oxygen balance, and varying the concentration and distribution of matrix embedded microcapsules provides spatiotemporal control. We demonstrate attenuation of hypoxia signaling in populations of stem cells, cancer cells, endothelial cells, and cancer spheroids. Capsule containing hydrogel films applied to chick chorioallantoic membranes encourage neovascularization, providing scope for topical treatments or hydrogel wound dressings. We further demonstrate versatility by loading capsules with ceria nanorods as "nanozymes" to modulate active oxygen species with potential as a cytoprotective treatment. The approach can be used in multiple formats, including deposition in hydrogels, as granular solids for 3D bioprinting, and as injectable biomaterials. Overall, this platforms simplicity and flexibility will prove useful for fundamental studies of oxygen-mediated processes in virtually any in vitro or in vivo format, with scope for inclusion in biomedical materials where controlling hypoxia may be clinically advantageous.

bioengineering↗

bioRxiv2020

Freeform printing of heterotypic tumor models within cell-laden microgel matrices

The tissue microenvironment is comprised of a complex assortment of multiple cell types, matrices, membranes and vessel structures. Emulating this complex and often hierarchical organization in vitro has proved a considerable challenge, typically involving segregation of different cell types using layer-by-layer printing or lithographically patterned microfluidic devices. Bioprinting in granular materials is a new methodology with tremendous potential for tissue fabrication. Here, we demonstrate the first example of a complex tumor microenvironment that combines direct writing of tumor aggregates, freeform vasculature channels, and a tunable macroporous matrix as a model to studying metastatic signaling. Our photocrosslinkable microgel suspensions yield local stiffness gradients between particles and the intervening space, while enabling the integration of virtually any cell type. Using computational fluid dynamics, we show that removal of a sacrificial Pluronic ink defines vessel-mimetic channel architectures for endothelial cell linings. Pairing this vasculature with 3D printing of melanoma aggregates, we find that tumor cells within proximity migrated into the prototype vasculature. Together, the integration of perfusable channels with multiple spatially defined cell types provides new avenues for modelling development and disease, with scope for fundamental research and drug development.

bioengineering↗

bioRxiv2019

Geometric regulation of histone state directs melanoma reprogramming

Malignant melanoma displays a high degree of cellular plasticity during disease progression, making classification of the heterogeneous population and selection of an appropriate therapy challenging. Signals in the tumor microenvironment are believed to influence melanoma plasticity through changes in the epigenetic state to guide dynamic differentiation and de-differentiation events that underlie tumorigenicity and dissemination. Here we uncover a relationship between geometric features at perimeter regions of multicellular melanoma aggregates, and reprogramming to a stem cell-like melanoma initiating cell (MIC) through histone marks H3K4Me2 and H3K9Ac. Using an in vitro tumor microengineering approach, we find concurrent expression of molecular MIC markers and spatial enrichment of these histone modifications at perimeter features. Chromatin immunoprecipitation and sequencing analysis demonstrates broad regulation of genes associated with SOX-, ETS-, and USF-families. SOX10 and PRDM14, transcriptional regulators with a putative role in several cancers, overlap with H3K9Ac and show elevated expression in cells along regions of perimeter curvature. siRNA knockdown of the epigenetic modifier PRDM14 abolishes the MIC phenotype suggesting a role in regulating melanoma heterogeneity. Our results suggest mechanotransduction at the periphery of melanoma tumors may orchestrate the activity of epigenetic modifiers to regulate histone state, cellular plasticity, and tumorigenicity.

cancer biology↗