Biology subjects

Baker, B. M.

Publications and source records attributed to Baker, B. M..

13 recordsLinked to original sources

bioRxiv2026

Beyond Histology: A Validated CUBIC-Based Workflow for Volumetric Analysis of Follicles and Cortical Vasculature in Human Ovarian Tissue

Research questionCan tissue clearing, combined with volumetric imaging, enable reliable, quantitative three-dimensional analysis of follicles and vasculature in intact human ovarian tissue? DesignA CUBIC-based clearing protocol was adapted for human ovarian medulla and cryopreserved cortex. Tissue from reproductive-aged donors was cleared, fluorescently labeled, and imaged using confocal and light sheet microscopy. Tissue expansion, imaging depth, and vascular morphometrics were quantified and follicle density was compared to conventional histology. ResultsClearing produced optically transparent tissue with a linear expansion factor of 1.2 across cortex and medulla. Imaging depth increased 6.5-11-fold in cortex and 6-8-fold in medulla. Follicle density measurements in immunolabeled cleared cortex were comparable to histology, supporting the validity of volumetric follicle quantification. Light sheet microscopy of lectin-labeled cortex revealed no significant donor-to-donor differences in vascular morphometrics, including mean vessel diameters of 12-14 {micro}m, branch point densities of 632-965 points/mm3, vessel length densities of 117-175 mm/mm3, and volume fractions of 1.9-2.3%. Volumetric imaging further illustrated heterogeneous spatial relationships between follicles and surrounding vessels. ConclusionTissue clearing and volumetric imaging complement routine histology and enable quantitative three-dimensional investigation of follicle-vascular interactions in intact human ovarian tissue, providing a framework for advancing fertility preservation and ovarian tissue transplantation research.

bioengineering↗

bioRxiv2025

HLA micropolymorphisms confine neoantigen conformational adaptability and guide T cell receptor selectivity

T cell receptor (TCR) restriction by highly polymorphic major histocompatibility complex (MHC) proteins is a foundation of cellular immunity. Although the effects of MHC polymorphisms on peptide binding and selection are well established, how micropolymorphisms within MHC supertypes impact immune recognition is poorly understood. Here, we identified a novel mechanism through which the micropolymorphisms in two closely related HLA-A3 superfamily members determine TCR specificity. We previously showed that TCRs specific for a public neoantigen from the PIK3CA oncogene presented by HLA-A*03:01 were unable to recognize the same epitope in the context of HLA-A*03:02. We found here that the micropolymorphisms distinguishing A*03:02 from A*03:01 exert their effect not by altering peptide binding or static structures, but by changing the conformational ensemble of the neoantigen in the groove, preventing it from adopting a conformation compatible with TCR binding. The effect is rooted in how the two polymorphic sites interact with other co-varying, evolutionarily coupled polymorphisms, reflecting a cross-groove network of interactions that controls the conformational adaptability of the peptide/HLA complex. We suggest polymorphism-dependent conformational adaptability reflects an evolved feature of class I MHC proteins that amplifies the impact of peptides in the groove, further diversifying epitopes and contributing to how TCRs and other immunoreceptors differentiate between antigens. Beyond this mechanistic insight, our findings emphasize the need for high-resolution HLA typing in efforts across immunology, including antigen-specific immunotherapy.

immunology↗

bioRxiv2025

Desmoplakin Haploinsufficiency Underlies Cell-Cell Adhesion Failure in DSP Cardiomyopathy and is Rescued by Transcriptional Activation

BackgroundTruncating variants in desmoplakin (DSPtv), are a leading cause of arrhythmogenic cardiomyopathy (ACM), often presenting with early fibrosis and arrhythmias disproportionate to systolic dysfunction. DSP is critical for cardiac mechanical integrity, linking desmosomes to the cytoskeleton to withstand contractile forces. While loss-of-function is implicated, direct evidence, both for DSP haploinsufficiency in human hearts and for the impact of mechanical stress on cardiomyocyte adhesion, has been limited, leaving the pathogenic mechanism unclear. MethodsWe analyzed explanted human heart tissue from patients with DSPtv (N=3), titin truncating variants (TTNtv, N=5), and controls (N=5) using RNA-sequencing and mass spectrometry. We generated human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) harboring patient-derived or CRISPR-Cas9 engineered DSPtv to model a range of DSP expression levels. Using a 2D cardiac muscle bundle (CMB) platform enabling live visualization of cell junctions, we developed an assay to assess cell-cell adhesion upon heightened contractile stress in response to the contractile agonist endothelin-1. CRISPR-interference (CRISPRi) was used to confirm the role of DSP loss, and CRISPR-activation (CRISPRa) was tested for therapeutic rescue. ResultsCompared to both control and TTNtv hearts, DSPtv human hearts exhibited reduced DSP at both the mRNA and protein level, as well as broadly disrupted desmosomal stoichiometry. Transcriptomic and proteomic analyses implicated cell adhesion, extracellular matrix, and inflammatory pathways. iPSC-CM models recapitulated DSP haploinsufficiency and desmosomal disruption. DSPtv CMBs showed normal baseline contractile function. However, they displayed marked cell-cell adhesion failure with contractile stress (75% failure vs. 8% in controls, p<0.001). Adhesion failure was prevented by the myosin inhibitor, mavacamten. CRISPRi-mediated DSP knockdown replicated this susceptibility to adhesion failure. Conversely, CRISPRa robustly increased DSP expression and rescued cell-cell adhesion failure in DSPtv CMBs (9% failure post-CRISPRa, p<0.001 vs. un-treated). Rescue occurred even when only the DSPII isoform was upregulated in a model with biallelic DSP transcript 1 loss of function. ConclusionsDSP haploinsufficiency is the major cause of DSP cardiomyopathy with a primary consequence of conferring vulnerability to cardiomyocyte cell-cell adhesion failure under heightened contractile stress. Transcriptional activation of DSP reverses this defect in preclinical models, establishing proof-of-concept for a potential therapeutic strategy in DSP cardiomyopathy.

genetics↗

bioRxiv2024

Clickable PEG-norbornene microgels support suspension bioprinting and microvascular assembly

The development of perfusable and multiscale vascular networks remains one of the largest challenges in tissue engineering. As such, there is a need for the creation of customizable and facile methods to produce robustly vascularized constructs. In this study, secondarily crosslinkable (clickable) poly(ethylene glycol)-norbornene (PEGNB) microbeads were produced and evaluated for their ability to sequentially support suspension bioprinting and microvascular self-assembly towards the aim of engineering hierarchical vasculature. The clickable PEGNB microbead slurry exhibited mechanical behavior suitable for suspension bioprinting of sacrificial bioinks, could be UV crosslinked into a granular construct post-print, and withstood evacuation of the bioink and subsequent perfusion of the patterned void space. Endothelial and stromal cells co-embedded within jammed RGD-modified PEGNB microbead slurries assembled into capillary-scale vasculature after secondary crosslinking of the beads into granular constructs, with endothelial tubules forming within the interstitial space between microbeads and supported by the perivascular association of the stromal cells. Microvascular self-assembly was not impacted by printing sacrificial bioinks into the cell-laden microbead support bath before UV crosslinking. Collectively, these results demonstrate that clickable PEGNB microbeads are a versatile substrate for both suspension printing and microvascular culture and may be the foundation for a promising methodology to engineer hierarchical vasculature.

bioengineering↗

bioRxiv2024

Distinct sets of molecular characteristics define tumor-rejecting neoantigens

Challenges in identifying tumor-rejecting neoantigens limit the efficacy of neoantigen vaccines to treat cancers, including cutaneous squamous cell carcinoma (cSCC). A minority of human cSCC tumors shared neoantigens, supporting the need for personalized vaccines. Using a UV-induced mouse cSCC model which recapitulated the mutational signature and driver mutations found in human disease, we found that CD8 T cells constrain cSCC. Two MHC class I neoantigens were identified that constrained cSCC growth. Compared to the wild-type peptides, one tumor-rejecting neoantigen exhibited improved MHC binding and the other had increased solvent accessibility of the mutated residue. Across known neoantigens that do not impact MHC binding, structural modeling of the peptide/MHC complexes indicated that increased solvent accessibility, which will facilitate TCR recognition of the neoantigen, distinguished tumor-rejecting from non-immunogenic neoantigens. This work reveals characteristics of tumor-rejecting neoantigens that may be of considerable importance in identifying optimal vaccine candidates in cSCC and other cancers.

immunology↗

bioRxiv2023

Manipulation of the nucleoscaffold potentiates cellular reprogramming kinetics

Somatic cell fate is an outcome set by the activities of specific transcription factors and the chromatin landscape and is maintained by gene silencing of alternate cell fates through physical interactions with the nuclear scaffold. Here, we evaluate the role of the nuclear scaffold as a guardian of cell fate in human fibroblasts by comparing the effects of transient loss (knockdown) and mutation (progeria) of functional Lamin A/C, a core component of the nuclear scaffold. We observed that Lamin A/C deficiency or mutation disrupts nuclear morphology, heterochromatin levels, and increases access to DNA in lamina-associated domains. Changes in Lamin A/C were also found to impact the mechanical properties of the nucleus when measured by a microfluidic cellular squeezing device. We also show that transient loss of Lamin A/C accelerates the kinetics of cellular reprogramming to pluripotency through opening of previously silenced heterochromatin domains while genetic mutation of Lamin A/C into progerin induces a senescent phenotype that inhibits the induction of reprogramming genes. Our results highlight the physical role of the nuclear scaffold in safeguarding cellular fate.

cell biology↗

bioRxiv2023

Single cell transcriptomic analysis of renal allograft rejection reveals novel insights into intragraft TCR clonality

Bulk analysis of renal allograft biopsies (rBx) identified RNA transcripts associated with acute cellular rejection (ACR); however, these lacked cellular context critical to mechanistic understanding. We performed combined single cell RNA transcriptomic and TCR/{beta} sequencing on rBx from patients with ACR under differing immunosuppression (IS): tacrolimus, iscalimab, and belatacept. TCR analysis revealed a highly restricted CD8+ T cell clonal expansion (CD8EXP), independent of HLA mismatch or IS type. Subcloning of TCR/{beta} cDNAs from CD8EXP into Jurkat76 cells (TCR-/-) conferred alloreactivity by mixed lymphocyte reaction. scRNAseq analysis of CD8EXP revealed effector, memory, and exhausted phenotypes that were influenced by IS type. Successful anti-rejection treatment decreased, but did not eliminate, CD8EXP, while CD8EXP were maintained during treatment-refractory rejection. Finally, most rBx-derived CD8EXP were also observed in matching urine samples. Overall, our data define the clonal CD8+ T cell response to ACR, providing novel insights to improve detection, assessment, and treatment of rejection.

immunology↗

bioRxiv2021

Mechanical intercellular communication via matrix-borne cell force transmission during vascular network formation

Intercellular communication is critical to the development and homeostatic function of all tissues. Previous work has shown that cells can communicate mechanically via transmission of cell-generated forces through their surrounding extracellular matrix, but this process is not well understood. Here, we utilized synthetic, electrospun fibrous matrices in conjunction with a microfabrication-based cell patterning approach to examine mechanical intercellular communication (MIC) between endothelial cells (ECs) during the assembly of microvascular networks. We found that cell force-mediated matrix displacements in deformable fibrous matrices underly directional migration of neighboring ECs towards each other prior to the formation of stable cell-cell connections. We also identified a critical role for intracellular calcium signaling mediated by focal adhesion kinase and TRPV4 during MIC that extends to multicellular assembly of vessel-like networks in 3D fibrin hydrogels. The results presented here are critical to the design of biomaterials that support cellular self-assembly for tissue engineering applications.

bioengineering↗

bioRxiv2021

Biomechanics and Myofibrillar Alignment Enhance Contractile Development and Reproducibility in Stem Cell Derived Cardiac Muscle

Human pluripotent stem cell derived cardiomyocytes (hPSC-CMs) allow novel investigations of human cardiac disease, but disorganized mechanics and immaturity of hPSC-CMs on two-dimensional (2D) surfaces have been hurdles for efficient and reproducible study of these cells. Here, we developed a platform of micron-scale 2D cardiac tissues (M2DCTs) to precisely control biomechanics in arrays of thousands of purified, independently contracting cardiac muscle strips in 2D. By defining geometry and workload in M2DCTs in this reductionist platform that does not incorporate other cell types, we show that myofibrillar alignment and auxotonic contractions at physiologic workload critically drive maturation of cardiac contractile function, calcium handling, and electrophysiology. Additionally, the organized biomechanics in this system facilitates rapid and automated extraction of contractile kinetic parameters from brightfield microscopy images, increasing the reproducibility and throughput of pharmacologic testing. Finally, we show that M2DCTs enable precise and efficient dissection of contractile kinetics in cardiomyopathy disease models.

cell biology↗

bioRxiv2021

Immunogenicity of a public neoantigen derived from mutated PIK3CA

Public neoantigens (NeoAgs) represent an elite class of shared cancer-specific epitopes derived from recurrent mutations in driver genes that are restricted by prevalent HLA alleles. Here, we report on a high-throughput platform combining single-cell transcriptomic and T cell receptor (TCR) sequencing to establish whether mutant (Mut) PIK3CA, among the most common genomically altered driver oncogenes, generates an immunogenic public NeoAg. Using this method, we developed a library of TCRs that recognize an endogenously processed neoepitope containing a common PIK3CA hotspot mutation that is restricted by HLA-A*03:01. Mechanistically, immunogenicity to this public NeoAg arises primarily from enhanced stability of the neopeptide/HLA complex caused by a preferred HLA anchor substitution. Structural studies indicated that the HLA-bound neopeptide presents a relatively "featureless" surface dominated by the peptides backbone. To overcome the challenge of binding such an epitope with high specificity and affinity, we discovered that a lead TCR clinical candidate engages the neopeptide through an extended interface aided by an unusually long {beta}-chain complementarity-determining region 3 (CDR3{beta}) loop. In a pan-cancer cohort of patients with diverse malignancies that express the PIK3CA public NeoAg, we observed spontaneous immunogenicity, NeoAg clonal conservation, and in a limited number of cases, evidence of targeted immune escape. Together, these results establish the immunogenic potential of Mut PIK3CA, creating a framework for off-the-shelf immunotherapies targeting this public NeoAg.

immunology↗

bioRxiv2021

Fiber density and matrix stiffness modulate distinct cell migration modes in a 3D stroma mimetic composite hydrogel

The peritumoral stroma is a complex 3D tissue that provides cells with myriad biophysical and biochemical cues. Histologic observations suggest that during metastatic spread of carcinomas, these cues influence transformed epithelial cells, prompting a diversity of migration modes spanning single cell and multicellular phenotypes. Purported consequences of these variations in tumor escape strategies include differential metastatic capability and therapy resistance. Therefore, understanding how cues from the peritumoral stromal microenvironment regulate migration mode phenotypes has prognostic and therapeutic value. Here, we utilize a synthetic stromal mimetic in which matrix fiber density and bulk hydrogel stiffness can be orthogonally tuned to investigate the contribution of these two key matrix attributes on MCF10A migration mode phenotypes, epithelial-mesenchymal transition (EMT), and invasive potential. We developed an automated computational image analysis framework to extract migratory phenotypes from fluorescent images and determine 3D migration metrics relevant to metastatic spread. Using this analysis, we find that matrix fiber density and bulk hydrogel stiffness distinctly contribute to a variety of MCF10A migration modes including amoeboid, single mesenchymal, multicellular clusters, and collective strands. Taking advantage of the tunability of this material platform, we identify a combination of physical and soluble cues that induces distinct heterogeneous migration modes originating from the same MCF10A spheroid and use this setting to examine a functional consequence of migration mode - apoptotic resistance. We find that cells migrating as part of collective strands are more resistant to staurosporine-induced apoptosis than either disconnected multicellular clusters or individual invading cells. Improved models of the peritumoral stromal microenvironment that help elucidate relationships between matrix attributes and cell migration mode can contribute to ongoing efforts to identify efficacious cancer therapeutics that address migration plasticity-based therapy resistances.

bioengineering↗

bioRxiv2020

Microengineered 3D pulmonary interstitial mimetics highlight a critical role for matrix degradation in idiopathic pulmonary fibrosis

Fibrosis is often untreatable and is characterized by aberrant tissue scarring from activated myofibroblasts. Although the extracellular matrix becomes increasingly stiff and fibrous during disease progression, how these physical cues impact myofibroblast differentiation in 3D is poorly understood. Here we describe a multicomponent hydrogel that recapitulates the 3D fibrous structure hallmark to the interstitial tissue regions where idiopathic pulmonary fibrosis (IPF) initiates. In contrast to findings on 2D hydrogels, myofibroblast differentiation in 3D was inversely correlated with hydrogel stiffness, but positively correlated with matrix fiber density. Employing a multi-step bioinformatics analysis of IPF patient transcriptomes and in vitro pharmacologic screening, we identify matrix-metalloprotease activity to be essential for 3D but not 2D myofibroblast differentiation. Given our observation that compliant degradable 3D matrices amply support fibrogenesis, these studies demonstrate a departure from the established relationship between stiffness and myofibroblast differentiation in 2D, and provide a new 3D model for studying fibrosis.

bioengineering↗

bioRxiv2019

Functional angiogenesis requires microenvironmental cues balancing endothelial cell migration and proliferation

Angiogenesis is a complex morphogenetic process that involves intimate interactions between multicellular endothelial structures and their extracellular milieu. In vitro models of angiogenesis can aid in reducing the complexity of the in vivo microenvironment and provide mechanistic insight into how soluble and physical extracellular matrix cues regulate this process. To investigate how microenvironmental cues regulate angiogenesis and the function of resulting microvasculature, we multiplexed an established angiogenesis-on-a-chip platform that affords higher throughput investigation of 3D endothelial cell sprouting emanating from a parent vessel through defined biochemical gradients and extracellular matrix. We found that two fundamental endothelial cell functions, migration and proliferation, dictate endothelial cell invasion as single cells vs. multicellular sprouts. Microenvironmental cues that elicit excessive migration speed incommensurate with proliferation resulted in microvasculature with poor barrier function and an inability to transport fluid across the microvascular bed. Restoring the balance between migration speed and proliferation rate rescued multicellular sprout invasion, providing a new framework for the design of pro-angiogenic biomaterials that guide functional microvasculature formation for regenerative therapies.

bioengineering↗