Biology subjects

Ranga, A.

Publications and source records attributed to Ranga, A..

14 recordsLinked to original sources

bioRxiv2025

SCAMPI: A scalable and accessible micropatterning platform for reproducible organoid morphogenesis

Micropatterning technologies enable biomimetic organoid generation with controlled in vitro morphogenesis but current approaches lack scalability. Here, we develop a mask-based photopatterning platform to enhance organoid culture reproducibility and scalability on diverse substrate formats. We demonstrate its use in generating architecturally defined neural organoids of varying geometries with reproducible morphogenesis. This technology provide a versatile and low-cost strategy for generating geometrically controlled organoids.

bioengineering↗

bioRxiv2025

Mesoscale landscaping of the TRIM protein family reveals a novel human condensatopathy

The mesoscale organization of cells is central to cellular physiology and pathology. Cellular condensates often form via biomolecular phase separation, mediated by intrinsically disordered regions (IDRs) and represent a key mechanism for mesoscale organization. The TRI-partite Motif (TRIM) family of ubiquitin ligases is implicated in diverse cellular functions and disease, yet the role of biomolecular condensation in TRIM family organization remains understudied. Here, we systematically investigate the mesoscale localization of 72 TRIM proteins, revealing that a majority form condensates in distinct cellular compartments. IDR content correlates with dynamic condensate formation, suggesting a critical role in mesoscale organization. Focusing on TRIM8, associated with a neuro-renal disorder, we demonstrate that disease-causing truncations of the TRIM8 C-terminal IDR result in a condensatopathy, characterized by disrupted condensation, proteasomal regulation, and TAK1/NF{kappa}B signaling. Functional assays in cellular and animal models link these disruptions to podocyte dysfunction and impaired response to injury. Our findings establish a framework for understanding condensatopathies and the mesoscale principles governing TRIM family organization and function.

cell biology↗

bioRxiv2024

Implantable 3D printed multiplexed microtunnels for spinal cord injury treatment

3D printed scaffolds offer a promising strategy for treating spinal cord injury (SCI). Here we present an innovative biotechnological approach for free-form 3D printing of scaffolds with a biomimetic architecture at a spatial resolution of up to a micrometer, designed for implantation in treatment of SCI in Wistar rats. The fabrication of scaffolds was based on 2-photon photopolymerization of organic polymers and was scalable to lesion geometries. The scaffolds were implemented as multiple densely packed squared parallel microtunnels (50 m per side) running their entire length. These microtunnels are separated by thin walls (5-10 m), rendering the scaffolds nearly hollow while maximizing their internal surface area. This design provides an optimal substrate, spatially aligned in the rostro-caudal direction, to support axonal and vascular ingrowth. We have found that the scaffolds, implanted in the excision of the lateral half-fragment of the spinal cord at the low thoracic level demonstrated excellent integration with surrounding tissue without the formation of a significant gliofibrous scar. Myelinated axons and oligodendrocytes, as well as vessels were observed in each microtunnel of the implanted scaffolds in 12 weeks after the operation with at least 1000 axons regenerating in the scaffold throughout its whole length. The treatment significantly improved motor function and reduced spasticity in the ipsilateral paretic limb by 8th week, with recovery sustained for at least 20 weeks. Thus, 3D oriented hollow scaffolds having a large internal surface area and direct continues microtunnels, effectively reducing axonal dispersion, mimic natural structure of the recipient tissue and create conditions for enhancing spinal cord regeneration and recovery of the motor function of the paretic limb.

bioengineering↗

bioRxiv2023

Force-mediated recruitment and reprogramming of healthy endothelial cells drive vascular lesion growth

Force-driven cellular interactions are known to play a critical role in cancer cell invasion, but have remained largely unexplored in the context of vascular abnormalities, partly due to a lack of suitable genetic and cellular models. One such vascular abnormality, cerebral cavernous malformation (CCM) is characterized by leaky, tumor-like vessels in the brain, where CCM mutant cells recruit wild-type cells from the surrounding endothelium to form mosaic lesions and promote lesion growth; however the mechanisms underlying this recruitment remain poorly understood. Here, we use 3D traction force microscopy in a in-vitro model of early angiogenic invasion to reveal that hyper-angiogenic CCM2-silenced endothelial cells enhance angiogenic invasion of neighboring wild-type cells through force and extracellular matrix-guided mechanisms. We show that mechanically hyperactive CCM2-silenced tips guide wild-type cells by exerting and transmitting pulling forces and by leaving degraded paths in the matrix as cues promoting invasion in a ROCKs-dependent manner. This transmission of forces is associated with a reinforcement of {beta}1 integrin-dependent adhesive sites and actin cytoskeleton in the wild-type followers. We also show that during this process wild-type cells are reprogrammed into stalk cells through activation of matrisome and DNA replication programs, eventually leading to cell proliferation. These observations unveil a novel vascular lesion growth mechanism where CCM2 mutants hijack the function of wild-type cells to fuel CCM lesion growth. By integrating biophysical computational methodologies to quantify cellular forces with advanced molecular techniques, we provide new insights in the etiology of vascular malformations, and open up avenues to study the role of cell mechanics in tissue heterogeneity and disease progression.

bioengineering↗

bioRxiv2022

Extracellular matrix flow guides in-vitro epithelial morphogenesis

Changes in spatial localization of the extracellular matrix (ECM) are necessary for establishing morphogenesis in multiple developmental contexts. Both ECM motion and tissue deformation require multicellular scale coordination, however the interplay between them remain largely unexplored. Here, we reveal a novel mechanism coupling morphogenetic events and epithelia-driven ECM flow in vitro. We show that exposure to ECM components triggers periodic morphogenesis of dome-shaped structures in human pluripotent stem cell (hPSC) monolayers, driven by directional ECM flow. We show that this flow is initiated by local symmetry breaking events, is driven by microvilli and requires unperturbed flow conditions, microvilli function, and cytoskeletal contractility. An in silico model shows that a reaction-diffusion-like mechanism is responsible for organizing local morphogenesis into global tissue-wide events. We validate this model by predicting changes in cell patterning landscape during mesoderm differentiation, and demonstrate changes in cellular identity by immunohistochemistry and scRNAseq. These results demonstrate that transport of ECM over epithelia, termed ECM flow, is a major contributor in sustaining morphogenesis and differentiation. Our in vitro approach suggests that ECM flow may be a broadly conserved mechanism guiding multi-cellular morphogenesis and may be further explored to investigate the role of ECM transport in other model systems.

bioengineering↗

bioRxiv2022

Local actuation of organoids by magnetic nanoparticles

Tissues take shape during development through a series of morphogenetic movements guided by local cell-scale forces. While current in vitro approaches subjecting tissues to homogenous stresses, it is currently no possible to recapitulate highly local spatially varying forces. Here we develop a method for local actuation of organoids using embedded magnetic nanoparticles. Sequential aggregation of magnetically labelled human pluripotent stem cells followed by actuation by a magnetic field produces localized magnetic clusters within the organoid. These clusters impose local mechanical forces on the surrounding tissue in response to applied global magnetic fields. We show that precise, spatially defined actuation provides short-term mechanical tissue perturbations as well as long-term cytoskeleton remodeling. We demonstrate that local magnetically-driven actuation guides asymmetric growth and proliferation, leading to enhanced patterning in human neural organoids. We show that this approach is applicable to other model systems by observing polarized patterning in paraxial mesoderm organoids upon local magnetic actuation. This versatile approach allows for local, controllable mechanical actuation in multicellular constructs, and is widely applicable to interrogate the role of local mechanotransduction in developmental and disease model systems.

developmental biology↗

bioRxiv2021

Engineering large-scale perfused tissues via synthetic 3D soft microfluidics

The vascularization of engineered tissues and organoids has remained a major unresolved challenge in regenerative medicine. While multiple approaches have been developed to vascularize in vitro tissues, it has thus far not been possible to generate sufficiently dense networks of small-scale vessels to perfuse large de novo tissues. Here, we achieve the perfusion of multi-mm3 tissue constructs by generating networks of synthetic capillary-scale 3D vessels. Our 3D soft microfluidic strategy is uniquely enabled by a 3D-printable 2-photon-polymerizable hydrogel formulation, which allows for precise microvessel printing at scales below the diffusion limit of living tissues. We demonstrate that these large-scale engineered tissues are viable, proliferative and exhibit complex morphogenesis during long-term in-vitro culture, while avoiding hypoxia and necrosis. We show by scRNAseq and immunohistochemistry that neural differentiation is significantly accelerated in perfused neural constructs. Additionally, we illustrate the versatility of this platform by demonstrating long-term perfusion of developing liver tissue. This fully synthetic vascularization platform opens the door to the generation of human tissue models at unprecedented scale and complexity.

bioengineering↗

bioRxiv2021

Paraxial mesoderm organoids model development of human somites

During the development of the vertebrate embryo, segmented structures called somites are periodically formed from the presomitic mesoderm (PSM), and give rise to the vertebral column. While somite formation has been studied in several animal models, it is less clear how well this process is conserved in humans. Recent progress has made it possible to study aspects of human paraxial mesoderm development such as the human segmentation clock in vitro using human pluripotent stem cells (hPSCs), however, somite formation has not been observed in these monolayer cultures. Here, we describe the generation of human paraxial mesoderm (PM) organoids from hPSCs (termed Somitoids), which recapitulate the molecular, morphological and functional features of paraxial mesoderm development, including formation of somite-like structures in vitro. Using a quantitative image-based screen, we identify critical parameters such as initial cell number and signaling modulations that reproducibly yielded somite formation in our organoid system. In addition, using single-cell RNA sequencing and 3D imaging, we show that PM organoids both transcriptionally and morphologically resemble their in vivo counterparts and can be differentiated into somite derivatives. Our organoid system is reproducible and scalable, allowing for the systematic and quantitative analysis of human spinal cord development and disease in vitro.

developmental biology↗

bioRxiv2021

Neuroepithelial organoid patterning is mediated by Wnt-driven Turing mechanism

Cell patterning in epithelia is critical for the establishment of tissue function during development. The organization of patterns in these tissues is mediated by the interpretation of signals operating across multiple length scales. How epithelial tissues coordinate changes in cell identity across these length scales to orchestrate cellular rearrangements and fate specification remains poorly understood. Here, we use human neural tube organoids as model systems to interrogate epithelial patterning principles that guide domain specification. In silico modeling of the patterning process by cellular automata, validated by in vitro experiments, reveal that the initial positions of floor plate cells, coupled with activator-inhibitor signaling interactions, deterministically dictate the patterning outcome according to a discretized Turing reaction-diffusion mechanism. This model predicts an enhancement of organoid patterning by modulating inhibitor levels. Receptor-ligand interaction analysis of scRNAseq data from multiple organoid domains reveals WNT-pathway ligands as the specific inhibitory agents, thereby allowing for the experimental validation of model predictions. These results demonstrate that neuroepithelia employ reaction-diffusion-based mechanisms during early embryonic human development to organize cellular identities and morphogen sources to achieve patterning. The wider implementation of such in vitro organoid models in combination with in-silico agent-based modeling coupled to receptor-ligand analysis of scRNAseq data opens avenues for a broader understanding of dynamic tissue patterning processes.

developmental biology↗

bioRxiv2021

Engineering neurovascular organoids with 3D printed microfluidic chips

The generation of tissues and organs requires close interaction with vasculature from the earliest moments of embryonic development. Tissue-specific organoids derived from pluripotent stem cells allow for the in vitro recapitulation of elements of embryonic development, however they are not intrinsically vascularized, which poses a major challenge for their sustained growth and for understanding the role of vasculature in fate specification and morphogenesis. Current organoid vascularization strategies do not recapitulate the temporal synchronization and spatial orientation needed to ensure in-vivo-like early co-development. Here, we developed a human pluripotent stem cell (hPSC)-based approach to generate organoids which interact with vascular cells in a spatially determined manner. The spatial interaction between organoid and vasculature is enabled by the use of a custom designed 3D printed microfluidic chip which allows for a sequential and developmentally matched co-culture system. We show that on-chip hPSC-derived pericytes and endothelial cells sprout and self-assemble into organized vascular networks, and use cerebral organoids as a model system to explore interactions with this de novo generated vasculature. Upon co-development, vascular cells interact with the cerebral organoid and form an integrated neurovascular organoid on chip. This 3D printing-based platform is designed to be compatible with any organoid system and is an easy and highly cost-effective way to vascularize organoids. The use of this platform, readily performed in any lab, could open new avenues for understanding and manipulating the co-development of tissue-specific organoids with vasculature.

bioengineering↗

bioRxiv2020

Valproic acid stimulates myogenesis in pluripotent stem cell-derived mesodermal progenitors in a Notch-dependent manner

Muscular dystrophies are debilitating neuromuscular disorders for which no cure exists. As this disorder affects both cardiac and skeletal muscle, patients would benefit from a cellular therapy that can simultaneously regenerate both tissues. The current protocol to derive bipotent mesodermal progenitors which can differentiate into cardiac and skeletal muscle relies on the spontaneous formation of embryoid bodies, thereby hampering further clinical translation. Additionally, as skeletal muscle is the largest organ in the human body, a high myogenic potential is necessary for successful regeneration. Here, we have optimized a protocol to generate chemically defined induced pluripotent stem cell-derived mesodermal progenitors (cdMiPs). We demonstrate that these cells contribute to myotube formation and differentiate into cardiomyocytes, both in vitro and in vivo. Furthermore, the addition of valproic acid, a clinically approved small molecule, increases the potential of the cdMiPs to contribute to myotube formation without compromising their ability to differentiate towards cardiomyocytes. This effect is mediated through the activation of the Notch signaling pathway. Taken together, these results constitute a novel approach to generate mesodermal progenitors with enhanced myogenic potential using clinically approved reagents, which opens the door to new therapeutic solutions in the treatment of muscular dystrophy.

cell biology↗

bioRxiv2020

Advanced in silico validation framework forthree-dimensional Traction Force Microscopy andapplication to an in vitro model of sproutingangiogenesis

In the last decade, cellular forces in three-dimensional hydrogels that mimic the extracellular matrix have been calculated by means of Traction Force Microscopy (TFM). However, characterizing the accuracy limits of a traction recovery method is critical to avoid obscuring physiological information due to traction recovery errors. So far, 3D TFM algorithms have only been validated using simplified cell geometries, bypassing image processing steps or arbitrarily simulating focal adhesions. Moreover, it is still uncertain which of the two common traction recovery methods, i.e., forward and inverse, is more robust against the inherent challenges of 3D TFM. In this work, we established an advanced in silico validation framework that is applicable to any 3D TFM experimental setup and that can be used to correctly couple the experimental and computational aspects of 3D TFM. Advancements relate to the simultaneous incorporation of complex cell geometries, simulation of microscopy images of varying bead densities and different focal adhesion sizes and distributions. By measuring the traction recovery error with respect to ground truth solutions, we found that while highest traction recovery errors occur for cases with sparse and small focal adhesions, our implementation of the inverse method improves two-fold the accuracy with respect to the forward method (average error of 23% vs. 50%). This advantage was further supported by recovering cellular tractions around angiogenic sprouts in an in vitro model of angiogenesis. The inverse method recovered more realistic traction patterns than the forward method, showing higher traction peaks and a clearer pulling pattern at the sprout protrusion tips.

bioengineering↗

bioRxiv2020

Actuation Enhances Patterning in Human Neural Tube Organoids

Tissues achieve their complex spatial organization through an interplay between gene regulatory networks, cell-cell communication, and physical interactions mediated by mechanical forces. Current strategies to generate in-vitro tissues have largely failed to implement such active, dynamically coordinated mechanical manipulations, relying instead on extracellular matrices which respond to, rather than impose mechanical forces. Here we develop devices that enable the actuation of organoids. We show that active mechanical forces increase growth and lead to enhanced patterning in an organoid model of the neural tube derived from single human pluripotent stem cells (hPSC). Using a combination of single-cell transcriptomics and immunohistochemistry, we demonstrate that organoid mechanoregulation due to actuation operates in a temporally restricted competence window, and that organoid response to stretch is mediated extracellularly by matrix stiffness and intracellularly by cytoskeleton contractility and planar cell polarity. Exerting active mechanical forces on organoids using the approaches developed here is widely applicable and should enable the generation of more reproducible, programmable organoid shape, identity and patterns, opening avenues for the use of these tools in regenerative medicine and disease modelling applications.

bioengineering↗

bioRxiv2020

A fully defined pluripotent stem cell derived multi-liver-cell model for steatohepatitis and fibrosis

Chronic liver injury, as observed in non-alcoholic steatohepatitis (NASH), progressive fibrosis, and cirrhosis, remains poorly treatable. Steatohepatitis causes hepatocyte loss in part by a direct lipotoxic insult, which is amplified by derangements in the non-parenchymal cellular (NPC) interactive network wherein hepatocytes reside, including, hepatic stellate cells, liver sinusoidal endothelial cells and liver macrophages. To create an in vitro culture model encompassing all these cells that allows studying liver steatosis, inflammation and fibrosis caused by NASH, we here developed a fully defined hydrogel microenvironment, termed hepatocyte maturation (HepMat) gel, that supports maturation and maintenance of pluripotent stem cell (PSC)-derived hepatocyte- and NPC-like cells for at least one month. The HepMat-based co-culture system modeled key molecular and functional features of TGF{beta}-induced liver fibrosis and fatty-acid induced inflammation and fibrosis better than monocultures its constituent cell populations. The novel co-culture system should open new avenues for studying mechanisms underlying liver steatosis, inflammation and fibrosis as well as for assessing drugs counteracting these effects.

bioengineering↗