Biology subjects

Gulati, N.

Publications and source records attributed to Gulati, N..

8 recordsLinked to original sources

bioRxiv2026

Apollo-IRE1: A Genetically Encoded Sensor for Live Cell and Multiplexed Imaging of ER Stress

Pancreatic beta cells face exceptional protein folding demands from high insulin production requirements, placing extraordinary stress on the ER and contributing to dysfunction in diabetes pathogenesis. Monitoring ER stress dynamics in living cells remains challenging due to the destructive nature of traditional biochemical methods and the limitations of existing fluorescent sensors. Here, we present Apollo-IRE1, a genetically encoded sensor that reports on stress-induced IRE1 oligomerization and associated change in homoFRET via changes in fluorescence anisotropy. Apollo-IRE1 provides a ratiometric, intensity-independent readout, resulting in low day-to-day variability and a minimal spectral bandwidth, enabling multiplexed imaging alongside other cellular parameters. Photobleaching and enhancement curve analysis show that Apollo-IRE1 exists in apparent monomeric, dimeric, and oligomeric states corresponding to baseline, moderate, and terminal ER stress conditions. The sensor also responds rapidly to chemical and physiological ER stressors in both immortalized beta-cell lines and primary mouse islet cells. These data establish Apollo-IRE1 as a practical tool for investigating ER stress dynamics in beta cells and other contexts where longitudinal single-cell measurements are essential.

bioengineering↗

bioRxiv2024

Development of an on-chip fluorescence anisotropy immunoassay for human C-peptide secretion reveals a general roadmap for tracer optimization

Fluorescence anisotropy immunoassays (FAIAs) are widely used to quantify the concentration of target proteins based on competition with a tracer in binding a monoclonal antibody. We recently designed an FAIA to measure mouse C-peptide secretion from living islets in a continuous-flow microfluidic device (InsC-chip). To develop an assay for human C-peptide, our initial selection of antibody-tracer pairings revealed the need to optimize both the dynamic range and the binding kinetics to measure the assay on-chip effectively. Here, we present strategies for developing an on-chip FAIA using two different monoclonal antibodies to achieve both a large dynamic range and high temporal resolution. The two monoclonal antibodies (Ab1 & Ab2) to human C-peptide initially showed low dynamic range and slow kinetics, preventing them from being used in an on-chip assay. To shorten the time-to-reach equilibrium for Ab1, we reengineered the tracer based on a comparison between the human and mouse C-peptide sequences, resulting in > 30-fold shorter time-to-reach equilibrium. To increase the relatively small dynamic range for Ab2, we used partial epitope mapping and targeted point mutations to increase the dynamic range by 45%. Finally, we validated both FAIAs by measuring depolarization-induced insulin secretion from individual hESC-islets in our InsC-chip. These strategies provide a general roadmap for developing FAIAs with high sensitivity and sufficiently fast kinetics to be measured in continuous-flow microfluidic devices.

bioengineering↗

bioRxiv2024

FAST-STEM: A human pluripotent stem cell engineering toolkit for rapid design-build-test-learn development of human cell-based therapeutic devices

Very recent clinical advances in stem cell derived tissue replacement and gene therapy, in addition to the rise of artificial intelligence-aided scientific discovery, have placed the possibility of sophisticated human cell-based therapies firmly within reach. However, development of such cells and testing of their engineered gene circuit components, has proven highly challenging, due to the need for generating stable cell lines for each design-build-test-learn engineering cycle. Current approaches to generating stable human induced pluripotent stem cell (hiPSC) lines are highly time-consuming and suffer from lack of control, poor integration efficiency, and limited functionality. Validation in clinically relevant stem cell derived tissues is also broadly lacking. Such drawbacks are prohibitive to repeatably conducting cutting-edge stem cell engineering with broad application within a realistic timeframe and will not scale with the future of regenerative medicine. We have developed FAST-STEM (Facile Accelerated Stem-cell Transgene integration with SynBio Tunable Engineering Modes), a hPSC engineering platform that drastically reduces the time to generate differentiation ready stem cell lines from several weeks to 5 days, exhibiting a ~612-fold improvement in transgene integration rate over previous methodologies. Additional FAST-STEM innovations include: (i) rapid and highly efficient transgene integration; (ii) copy number control; (iii) simultaneous or consecutive integration of multiple gene cassettes; (iv) library screen capability. In addition to this unique functional versatility, platform transportability and broad use case for stem cell-engineering was confirmed by differentiation into eight different cell types across nine different laboratories. This platform dramatically lowers the bar for integration of synthetic biology with regenerative medicine, enabling experiments which were previously deemed logistically impossible, thus paving the way for sophisticated human cell device development.

bioengineering↗

bioRxiv2024

A cryopreservation strategy for myoblast storage in paper-based scaffolds for inter-laboratory studies of skeletal muscle health

Three-dimensional tissue-engineered models are poised to facilitate understanding of skeletal muscle pathophysiology and identify novel therapeutic agents to improve muscle health. Adopting these culture models within the broader biology community is a challenge as many models involve complex methodologies and significant investments of time and resources to optimize manufacturing protocols. To alleviate this barrier, we developed a protocol with commercially available reagents to cryopreserve myoblasts in a 96-well compatible format that allows tissues to be transferred to users without expertise in 2D or 3D skeletal muscle cell culture. We validate that myoblasts encapsulated in a hydrogel and cryopreserved in paper-based scaffolds maintain cell viability, differentiation, and function via acetylcholine-induced transient calcium responses. Furthermore, we demonstrate successful shipping of myoblasts cryopreserved in paper-based scaffolds to intra-provincial and international collaborators who successfully thawed, cultured, and used the 3D muscle tissues. Finally, we confirm the application of our method to study muscle endogenous repair by seeding freshly isolated skeletal muscle stem cells to cryopreserved then differentiated and injured tissues, demonstrating expected responses to a known stimulator of muscle stem cell self-renewal, p38/{beta} MAPKi. Altogether, our 3D myoblast cryopreservation protocol offers broadened access of a complex skeletal muscle tissue model to the research community.

bioengineering↗

bioRxiv2023

Culture substrate stiffness impacts human myoblast contractility-dependent proliferation and nuclear envelope wrinkling

The remarkable self-repair ability of skeletal muscle tissues is driven by muscle stem cells, whose activities are orchestrated by a variety of transient cues present in their microenvironment. Understanding how the cross-interactions of these biophysical and biochemical microenvironmental cues influences muscle stem cells and their progeny is crucial in strategizing remedies for pathological dysregulation of these cues in aging and disease. In this study, we investigated the cell-level influences of extracellular matrix ligands in the context of mechanically tuned culture substrates on primary human myoblast contractility and proliferation within 16 hours of plating. We found that fibronectin led to stronger stiffness-dependent responses in myoblasts compared to laminin and collagen as revealed by cell spreading area, focal adhesion morphology, traction stresses, and proliferation. While we did not observe stiffness-dependent changes in cell fate signatures at this early time-point, an analysis of the stiffness-sensitive myoblast proteome uncovered an enrichment for differentiation associated signatures on 17 kPa culture substrates, in addition to distinct profiles with relation to metabolism, cytoskeletal regulation, ECM interactions, and nuclear component enrichment. Interestingly, we found that softer substrates increased the incidence of myoblasts with a wrinkled nucleus, and that the extent of wrinkling could predict Ki67 expression. While nuclear wrinkling and Ki67 expression could be controlled by pharmacological manipulation of cellular contractility, cellular traction stresses did not correlate with nuclear envelope wrinkling level. These results provide new insights into the regulation of human myoblast stiffness-dependent contractility response by ECM ligands and also highlight a link between myoblast contractility and proliferation.

bioengineering↗

bioRxiv2023

A Flexible Transgene Integration Landing-Pad Toolkit in Human Induced Pluripotent Stem Cells Enables Facile Cellular Engineering, Gene Zygosity Control, and Parallel Transgene Integration

Development of a repeatable method for delivering transgene payloads to human induced pluripotent stem cells (hiPSCs) without risking unintended off-target effects is not fully realized. Yet, such methods are indispensable to fully unlocking the potential for applying synthetic biological approaches to regenerative medicine, delivering quantum impacts to cell-based therapeutics development. Here we present a toolkit for engineering hiPSCs centred on the development of two core landing-pad cell-lines, facilitating rapid high-efficiency delivery of transgenes to the AAVS1 safe-harbour locus using the Bxb1 large-serine recombinase. We developed two landing-pad cell lines expressing green and red fluorescent reporters respectively, both retaining stemness whilst fully capable of differentiation into all three germ layers. A fully selected hiPSC population can be isolated within 1-2 weeks after landing-pad recombinase-mediated cassette exchange. We demonstrate the capability for investigator-controlled homozygous or heterozygous transgene configurations in these cells. As such, the toolkit of vectors and protocols associated with this landing-pad hiPSC system has the potential to accelerate engineering workflows for researchers in a variety of disciplines.

synthetic biology↗

bioRxiv2023

mini-MEndR: A miniaturized 96-well predictive assay to evaluate muscle stem cell mediated repair

Functional evaluation of molecules that are predicted to promote stem cell mediated endogenous repair often requires in vivo transplant studies that are low throughput and hinder the rate of discovery. Here, we offer a strategy to rapidly test and prioritize molecules for functional validation studies. We miniaturized, simplified and expanded the functionality of a previously developed muscle endogenous repair (MEndR) in vitro assay that was shown to capture significant events of the first week of the in vivo muscle endogenous repair process. The new "mini-MEndR assay" consists of miniaturized cellulose scaffolds designed to fit in 96-well plates. The scaffold pores are infiltrated with myoblasts encapsulated in a fibrin-based hydrogel to form thin, engineered skeletal muscle tissues. By evaluating multiple commercially available human primary myoblast lines in 2D and 3D culture, we establish quality assurance metrics for cell line selection that standardize myotube template quality. Pre-adsorbing thrombin to the cellulose scaffolds facilitates in situ tissue polymerization, a critical modification that enables users proficient in myoblast culture to rapidly acquire myotube template fabrication expertise. Following the generation of the 3D myotube template, muscle stem cells (MuSCs), enriched from digested mouse skeletal muscle tissue using an improved magnetic-activated cell sorting protocol, are engrafted onto the engineered human muscle template. A regenerative milieu is then introduced by injuring the muscle tissue with a myotoxin. Addition of a known modulator of MuSC mediated repair recapitulates the in vivo outcomes (enhanced muscle production and Pax7+ cell expansion), but only in the presence of both the stem cells and the regenerative milieu. By fluorescently labeling the mouse MuSCs, we demonstrate the feasibility of co-evaluating human and mouse Pax7+ cell responses to drug treatment, thereby expanding the utility of the assay. Importantly, phenotypic data is collected with a high-content imaging system and is analyzed using CellProfiler-based image analysis pipelines. The miniaturized predictive assay offers a simple, scaled platform with which to co-investigate human and mouse skeletal muscle endogenous repair molecular modulators, and thus is a promising strategy to accelerate the muscle endogenous repair discovery pipeline.

bioengineering↗

bioRxiv2021

Saturation variant interpretation using CRISPR prime editing

Over the last decade, next generation sequencing has become widely implemented in clinical practice. However, as genetic variants of uncertain significance (VUS) are frequently identified, the need for scaled functional interpretation of such variants has become increasingly apparent. One method to address this is saturation genome editing (SGE), which allows for scaled multiplexed functional assessment of single nucleotide variants. The current applications of SGE, however, rely on homology-directed repair (HDR) to introduce variants of interest, which is limited by low editing efficiencies and low product purity. Here, we have adapted CRISPR prime editing for SGE and demonstrated its utility in understanding the functional significance of variants in the NPC1 gene underlying the lysosomal storage disorder Niemann-Pick disease type C1 (NPC). Additionally, we have designed a genome editing strategy that allows for the haploidization of gene loci, which permits isolated variant interpretation in virtually any cell type. By combining saturation prime editing (SPE) with a clinically relevant assay, we have functionally scored and interpreted 256 variants in NPC1 haploidized HEK293T cells. To further demonstrate the applicability of this strategy, we used SPE and cell model haploidization to functionally score 465 variants in the BRCA2 gene. We anticipate that our work will be translatable to any gene with an appropriate cellular assay, allowing for more rapid and accurate diagnosis and improved genetic counselling and ultimately precise patient care.

genetics↗