Everardo Anta Galvan, DVM, MS – The effect of pluripotency state asynchrony on the developmental potential of porcine complementation chimeras
Tanja Dominko lab, Department of Surgery
Abstract: Interspecies blastocyst complementation (IBC) utilizing human induced pluripotent stem cells (iPSCs) offers an alternative to current xenotransplantation strategies that rely on xenoantigen-edited pigs. By generating human iPSC-derived organs within organ-deficient pig embryos, IBC has the potential to produce chimeric organs with improved immunological compatibility. However, blastocyst-stage complementation with human iPSCs yields low chimerism, often below 2%, reflecting poor human donor cell survival and limited contribution to the developing embryo. We hypothesize that complementation at the 4-cell stage, coinciding with the onset of porcine embryonic genome activation (EGA), will provide a more permissive environment for synchronization of iPSC pluripotency state with that of the developing embryo, thereby enhancing human iPSC survival, integration, and lineage contribution. To establish parameters for future human-porcine chimeras, we characterized preimplantation integration dynamics of porcine iPSCs microinjected into EGA-stage parthenotes. We evaluated chimerism and viability via time-lapse imaging, nuclear staining, and TUNEL assays (n=44). iPSC proliferation positively correlated with host blastomere number (P = 0.005), and embryos reaching the blastocyst stage contained significantly more iPSCs than those undergoing developmental arrest (P < 0.05). Apoptosis rates were not different between iPSCs and host blastomeres (P = 0.75). These findings identified the EGA stage as a receptive and supportive environment for complementation and showed that donor cell persistence is linked to continued embryo development rather than donor cell viability alone. Although we used intraspecies complementation, the data indicate that asynchrony in pluripotency state (primed iPSCs are not outperformed by the totipotent/naïve state of embryonic blastomeres) does not affect iPSC or preimplantation embryo survival. Future complementation of ETV2-null host embryos with human iPSCs will investigate the feasibility of generating humanized, immunologically compatible vascular tissues for xenotransplantation.
Srishti Goswami, MS – Establishing Human Stem Cell Derived Retinal Organoids as a robust model to study blindness disorders and develop therapeutics
Raunak Sinha Lab, Department of Ophthalmology and Visual Sciences
Abstract: Human pluripotent stem cell (hPSC)-derived retinal organoids (ROs) represent a transformative advancement, offering a 3D system to recapitulate the complexity of the developing human retina. They serve as robust platforms for modelling inherited retinal diseases and testing therapies. The ability of RO photoreceptors to successfully relay sensory information depends on the formation of functional synapses to facilitate downstream signalling between photoreceptors and second-order neurons. Thus, validating the expression and function of photoreceptor synaptic proteins is critical. One family of synaptic proteins that facilitates neurotransmitter release from photoreceptor terminals are calcium channels but their physiological properties and expression profiles of these channels within RO photoreceptors remains unknown. This information is required as a key step for establishing ROs as authentic models of human retina.
We employed patch-clamp electrophysiology to measure calcium currents from RO photoreceptors and characterized the functional state of the organoid cone photoreceptors. Cone photoreceptors in primate retina support bright light and colour vision; mutations in their calcium channels lead to vision disorders. We benchmarked our findings of RO cone photoreceptor calcium channel properties against a gold-standard reference of fetal and adult macaque cone photoreceptors.
Our findings indicate that a subset of organoid cone photoreceptors exhibit synaptic function similar to fetal macaque. We are also planning to compare the trajectory of calcium channel functional profiles across RO photoreceptor developmental timepoints, correlating these with their light response profile. By aligning physiological profiles of RO photoreceptors across development with primate retinal physiology, this work establishes a rigorous framework for assessing the authenticity of lab-generated ROs. These findings are significant for generating disease-relevant models for therapeutic screening and drug discovery.
Hannah Hwang, BS – Promoter-proximal pausing establishes bioenergetic readiness in quiescent muscle stem cells
Roméo Blanc Lab, Department of Cell and Regenerative Biology
Abstract: Skeletal muscle regeneration relies on a rare population of muscle stem cells (MuSCs), which preserve their regenerative capacity by entering quiescence, a reversible state of growth arrest. These quiescent muscle stem cells face a fundamental paradox: transcription must remain restrained to maintain quiescence, yet gene expression must be rapidly activated upon injury. However, the mechanisms that enable this balance remained poorly understood. Here, we show that quiescence represents a state of strategic pause, in which transcriptional pausing maintains genes in readiness for rapid activation following injury.
Using Precision Run-On sequencing (PRO-seq), we found that quiescent MuSCs exhibit widespread genome-wide promoter-proximal transcriptional pausing, which is broadly released upon activation. Gene Ontology analysis of paused genes revealed strong enrichment for mitochondrial pathways. Remarkably, ranking mitochondrial genes by pausing index showed that nearly 80% fall into a transcriptionally poised category, suggesting that energy production programs are strategically restrained in quiescence. Transcription factor enrichment analysis identified the YY1 as the top predicted regulator of these paused gene set. Consistent with this, CUT&Tag profiling revealed that these paused genes carry a dual epigenetic signature of H4K20me1 and H4K16ac in quiescence, with selective loss of H4K16ac upon activation. These findings suggest that dynamic histone modifications cooperate with transcriptional regulators to stabilize promoter-proximal pausing at metabolic genes.
Together, our findings uncover a previously unrecognized regulatory layer in which quiescent MuSCs selectively enforce transcriptional pausing at mitochondrial gene programs to enable a rapid surge of energy production upon activation. These findings reveal a regulatory mechanism that preserves metabolic readiness in quiescent MuSCs and suggest new avenues for enhancing muscle regeneration in aging and degenerative muscle diseases.
Susmit Mhatre, MS – A potential asymmetrically dividing vimentin expressing progenitor population in adult mouse hippocampus
Darcie L. Moore Lab, Department of Neuroscience
Abstract: Adult neural stem cells (NSC) are primarily quiescent and lose their ability to exit quiescence with aging and disease. We have previously found in vitro that the intermediate filament vimentin collapses around aggresome during quiescence exit to forming a vimentin cage. As the NSC divides, it asymmetrically segregates these cellular cargoes to one daughter cell, leading to a decrease in its proliferation rate while the other daughter remains able to rejuvenate the niche. In the absence of vimentin, NSCs have a reduced ability to exit quiescence. Although the role of vimentin in quiescence exit has been shown in vitro, its function in a physiological setting in vivo remains unknown. To study how vimentin is regulated in vivo, we developed a novel endogenous Vimentin-mScarlet reporter mouse line and crossed with Nestin-GFP. Single-cell RNA sequencing and prospective FACS isolation of distinct cell populations (mScarlet+ and/or GFP+ cells) revealed that Vimentin expression defines a downstream progenitor stage within the adult neurogenic niche. In primary and secondary sphere assays, Vim-mScar+ positive progenitors formed significantly smaller spheres and displayed reduced self-renewal compared to Nestin-GFP+ NSCs, which upregulate vimentin upon proliferation. Longitudinal single-cell timelapse imaging further revealed distinct division kinetics: while Nestin-GFP+ NSCs undergo predominantly symmetric divisions, Vim-mScar+ progenitors undergo asymmetric fate divisions. Differentiation assays following growth factor withdrawal showed that Vim-mScar+ progenitors exhibit significantly reduced neuronal potential and are strongly biased toward astrogliogenesis. We currently have establish capabilities for longitudinal live two-photon imaging paired with inducible lineage-tracing mouse lines, offering a robust platform to directly resolve these progenitor division kinetics and fate behaviors in vivo. Together, these findings potentially report vimentin as a key marker for an asymmetrically dividing glial progenitor population in the adult hippocampus, expanding our understanding of lineage commitment in adult neurogenesis.
Sayandeep Saha, MS – Hypoimmune ICAM-1 knockout human pluripotent stem cell-derived endothelial cells evade innate and adaptive immune responses under physiological conditions
Matthew E. Brown Lab, Department of Surgery
Abstract: Immune-mediated rejection remains a major barrier to the clinical translation of human pluripotent stem cell (PSC)-derived cellular therapies, with endothelial cells (ECs) serving as early and critical targets of host immunity in vascular grafts. Current hypoimmune strategies primarily rely on major histocompatibility complex (MHC) disruption to blunt T cell recognition but can provoke NK cell missing-self responses and are not mechanistically targeted to prevent innate immune mechanisms, particularly neutrophil-driven inflammatory injury that dominates the earliest stages of graft rejection. We recently identified intercellular adhesion molecule-1 (ICAM-1) as a promising hypoimmune target that reduces leukocyte adhesion and T cell activation in static 2D culture while preserving MHC expression and avoiding NK cell missing-self recognition. To advance this strategy toward clinical relevance, here we investigate ICAM-1 knockout (KO) PSC-derived ECs under physiologically relevant conditions that model early inflammatory events following transplantation. ICAM-1 KO PSC-ECs retained endothelial identity and function, including acetylated LDL uptake, tube formation, nitric oxide production, engraftment kinetics comparable to wild-type controls in vivo, and the ability to form perfusable 3D vascular lumens that recapitulate native vessel geometry and microenvironmental organization. Under shear flow, ICAM-1 deletion markedly reduced neutrophil adhesion to the vasculature. In complementary static and co-culture assays, loss of ICAM-1 significantly diminished neutrophil-mediated oxidative stress, consistent with impaired immune–endothelial engagement. In 2D static and 3D vascular lumen models, ICAM-1 KO endothelium profoundly suppressed neutrophil adhesion and transendothelial migration without compromising barrier integrity. Together, these findings establish ICAM-1 as a central adhesion checkpoint governing early innate immune injury. We demonstrate that ICAM-1 knockout enables the generation of structurally intact, functionally competent, and hypoimmune human vasculature under physiological conditions, offering a broadly applicable strategy to improve early graft survival in PSC-based vascular and other regenerative cell therapies.
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