DNA repair in biochemical and genetic experimental systems permits a precise definition of enzyme requirements and mechanistic steps. Comparing these findings to repair events at naturally occurring damage sites in multicellular organisms is essential for confirming and expanding these insights into a physiologic context. However, heterogeneity in any normal cell population increases with each cell division, and the reliable detection of replication-independent DNA damage sites and their repair has been a major barrier. Here, we examine single human colon crypts, which harbor natural cell clones, using a novel whole-genome sequencing (WGS) method to identify complex insertion-deletion (indel) in the crypt stem cells. Analysis of complex indel events likely repaired by non-homologous end joining occurring in crypt stem cells permits inferences about the in vivo repair of naturally occurring DNA damage within physiologically-relevant chromatin in normal human cells.

Parkin, an E3 ubiquitin ligase encoded by PARK2 , plays a key role in both hereditary and sporadic Parkinson's disease (PD), yet there are no therapies currently available that can target this important pathway. Here, we show that Parkin is critical for successful neuronal differentiation and survival, and we develop small-molecule Parkin agonists that can protect dopaminergic neurons. Upon differentiation of neural progenitor cells, loss of Parkin results in a reduced capacity to maintain neuronal cell state, dopaminergic neuronal phenotypes, and stress resistance. Moreover, Parkin loss disrupted cell morphology and the stability of neurites. Transcriptional and single-cell analyses reveal that Parkin controls critical pathways regulating stem-like cell transitions and is needed for stable neuronal maturation. We also examined the effects of FB231, a small molecule enhancer of Parkin E3 ligase activity, in models of PD. FB231 reduced pathological α-synuclein and enhanced cell survival in human iPSC-derived dopaminergic neurons treated with α-synuclein preformed fibrils. Furthermore, FB231 attenuated α-synuclein pathology and dopaminergic neurodegeneration in a gut α-synuclein murine model of PD. Our findings support that Parkin plays a crucial role in maintaining neuronal homeostasis and that pharmacologic activation of Parkin may be a promising strategy to attenuate neurodegeneration in PD.

Filamentation induced by cAMP domain-containing protein (FICD) is an endoplasmic reticulum (ER)-resident adenylyltransferase that catalyzes protein AMPylation, a post-translational modification. Although FICD-mediated AMPylation has been linked to the fine-tuning of proteostasis and neuronal integrity, its role in neurodegenerative diseases characterized by protein dyshomeostasis remains unclear. Parkinson's disease (PD) is defined by dopaminergic neurodegeneration and aggregation of α-synuclein (aSyn) as a consequence of impaired protein homeostasis. We therefore investigated whether dysregulated FICD-mediated AMPylation contributes to PD pathogenesis.

Centromeres ensure proper chromosome segregation during cell division, yet the organization and regulation of centromeric chromatin within satellite DNA arrays remain incompletely understood. Here, we leverage the complete diploid human genome benchmark (T2T-HG002) to provide a detailed study of centromeric sequence and chromatin architecture on individual haplotypes. Using adaptive-sampling-enriched, ultra-long-read DiMeLo-seq, we achieve single-molecule chromatin profiling across all centromeres, revealing that along single chromatin fibers, CENP-A, the histone variant specifying centromere identity, forms multiple discrete subdomains within hypomethylated centromere dip regions (CDRs) that are flanked by H3K9me3-enriched heterochromatin. Despite underlying sequence variation, CDRs localize to sequence-homogeneous domains and maintain relatively balanced CENP-A dosage and aggregate length across all chromosomes and between haplotypes. Further, we show that bidirectional changes to centromeric and pericentromeric DNA methylation are accompanied by changes to centromeric chromatin architecture. In passaged cells with centromeric hypomethylation, subdomain boundaries are eroded, and adjacent CENP-A domains tend to merge and expand. Conversely, in pluripotent stem cells with centromeric hypermethylation, CDRs are fundamentally reorganized, such that discrete hypomethylated domains are frequently consolidated into broader contiguous tracts. These methylation-associated CDR restructuring events suggest that DNA methylation acts as a principal regulator of human centromere organization, with implications for understanding centromere plasticity, epigenetic inheritance, and chromosomal instability in development and disease.

Chimeric antigen receptor (CAR) T cell therapy has transformed the treatment of B cell malignancies, but translation to acute myeloid leukemia (AML) has been hindered by on-target, off-tumor (OTOT) toxicity. In particular, endothelial cell (EC)-specific toxicity has limited clinical translation of promising leukemia stem cell-enriched targets such as CD93. Innovative strategies to mitigate EC damage while preserving antileukemic efficacy are needed.

Astrocytes play a key role in neuronal homeostasis and in various neural disorders. The generation of astrocytes from neural progenitor cells (NPCs) and its functions are under a complex control of several signaling networks and transcription factors. In this study, we demonstrate that the transcription factor, GLIS similar 3 (GLIS3), which has been implicated in several neurodegenerative diseases, is highly expressed in astrocytes, and is required for the efficient differentiation of human NPCs into astrocytes. Loss of GLIS3 function greatly impairs astrocytes differentiation, resulting in reduced expression of astrocyte markers, whereas expression of exogenous GLIS3 restores the induction of astrocyte specific genes indicating a critical role for GLIS3 in astrocyte differentiation. Integrated transcriptomic and cistromic analyses revealed that GLIS3 directly regulates the transcription of several astrocyte-associated genes, including GFAP , SLC1A2 , NFIA , and ATF3 , in coordination with lineage-determining factors, such as STAT3, NFIA, and SOX9. We hypothesize that GLIS3 dysfunction disrupts this transcriptional network thereby contributing to astrocyte-associated neurological disorders. Identification of GLIS3 as a key regulator of astrocyte differentiation and gene expression will advance our understanding of its role in neurodegenerative diseases and may provide a new therapeutic target.

Med12 and Med13 are components of the kinase module of the mediator complex. Mutations of Med12 and Med13 have been associated with neurodevelopmental disorders and various cancers. However, their functions in neural development are not well understood. Here we show that in the developing Drosophila brain, Med12 and Med13 are required to prevent tumorigenic dedifferentiation of intermediate neural progenitors (INPs) and maintain neural stem cell (NSC) self-renewal. We further demonstrate that Med12 and Med13 prevent INP dedifferentiation by coordinating with a subset of core mediator complex subunits to mediate the activation of genes required for INP fate commitment. In contrast, during the maintenance of NSC self-renewal, Med12 and Med13 antagonize the function of a different subset of core mediator complex subunits. Together, our findings reveal that Med12 and Med13 perform two distinct functions in neural progenitors by coordinating with one subset of core mediator complex subunits while antagonizing another.

Menin scaffolds the oncogenic histone-lysine-N-methyltransferase (KMT2A)-fusion protein (FP) complex in KMT2A-r and wild-type KMT2A complex in NPM1-m acute myeloid leukemia (AML). Menin inhibitors (MIs) are effective in KMT2A-r AML and NPM1-m AML. However, not all patients respond to MIs as monotherapy. In this preclinical study, we demonstrate that the MI ziftomenib, in combination with the XPO1 inhibitor selinexor, synergistically inhibited the growth of multiple KMT2A-r and NPM1-m AML cell lines (CI<1). The combination suppressed colony formation in primary CD34+ KMT2A-r progenitor cells without affecting normal stem cells. Robust apoptosis and decreased G2/M populations were also evident. The combination downregulated HOXA9 and MEIS1 while upregulating monocytic differentiation marker CD11b in both the AML molecular signatures. RNA sequencing and proteomic analysis in KMT2A-r revealed suppression of multiple bona fide menin-KMT2A target genes. Our mechanistic studies also identified a novel role of XPO1 in stabilizing menin's binding to chromatin and its interactions with KMT2A and KMT2A/MLLT3. XPO1 inhibitor-mediated disruption of these interactions, particularly in combination with ziftomenib, synergistically impairs oncogenic transcriptional programs. In vivo, combination therapy improved survival in both MV4;11 and OCI-AML3 cell line and primary patient-derived KMT2A-r and NPM1-m AML xenograft models in NSG mice, effective even at reduced drug doses. These preclinical findings demonstrate that simultaneous inhibition of the menin-KMT2A interaction and XPO1 can be a more effective translational strategy for treating KMT2A-r and NPM1-m AML than MI monotherapy to deepen responses and delay/prevent relapses.

Critical sized craniomaxillofacial bone defects do not heal naturally and often exhibit chronic inflammatory responses that restrict regeneration. It is increasingly apparent that biomaterials must facilitate dynamic crosstalk between immune cells, such as macrophages, and osteoprogenitors to resolve inflammation and accelerate regeneration. Here, we evaluate interactions between macrophages in a neutral (M0) or pro-inflammatory (M1) state with mesenchymal stem cells (MSCs) in a basal or licensed state within a mineralized collagen scaffold. We reveal that MSC-macrophage crosstalk influences significant changes in osteoprogenitor cell differentiation and immune cell polarization. Notably, crosstalk between MSCs and macrophages drives an early-stage inflammatory response, which enhances the immunomodulatory activity of MSCs via secretion of IL-6, an effect that is heightened for already licensed MSCs. The presence of macrophages in the co-cultures upregulated osteogenic (ALPL, BMP2, COL1A2, and RUNX2) and angiogenic genes (ANGPT1) in basal MSC groups. Further, MSC-macrophage interactions subsequently drive increased M2-like macrophage polarization as early as 7 days of culture, as indicated by surface marker expression. These findings show that biomaterial scaffolds can be leveraged as mediators of MSC-mediated immunomodulation with an emphasis on achieving early-stage pro-inflammatory phenotypes that drive subsequent macrophage polarization and markers of increased regenerative potency.

Neuromodulators influence critical functions of the developing human brain and regulate behavioral states. Dysfunction of neuromodulatory systems is often involved in neuropsychiatric disease and many drugs for these conditions act on these signaling pathways. Recent advances in stem cell biology have made it possible to derive a wide range of cells across the developing human nervous system in regionalized organoids and to functionally integrate them into assembloids, however they currently do not systematically incorporate neuromodulation. Here, we generated human midbrain-hindbrain organoids (hMHO) from human induced pluripotent stem (hiPS) cells and fused them with human cortical organoids (hCO) to form neuromodulatory assembloids (hNMA). We focus on serotonin (5-hydroxytryptamine, 5-HT) as one key neuromodulator and found characteristic gene expression patterns and electrophysiological properties of serotonergic neurons (5-HT neurons) in the hMHO. In hNMA, 5-HT neurons projected into hCO, released 5-HT and modulated cortical network activity. To explore the applicability of this system in human disease, we studied 22q11.2 deletion syndrome (22q11.2DS), a common microdeletion associated with high risk for neuropsychiatric disease and defects in 5-HT signaling. We found aberrant 5-HT dynamics in hNMA from patient hiPS cell lines that were rescued by administration of a selective serotonin reuptake inhibitor (SSRI). Taken together, hNMA can be used to study human 5-HT dynamics and uncover disease phenotypes which could facilitate therapeutic development.

Critical-sized craniomaxillofacial (CMF) defects affect the skull, face, and jaw, arising from conditions such as cleft palate, oncologic resections, and high energy impacts, and due to their large size and irregular geometry, cannot heal naturally by the body, thus requiring surgery. The field of biomedical research has long recognized the need to develop higher order biomaterial model systems for improved disease characterization and translational therapeutic/material progress. There is, however, difficulty in developing these workflows at the scale of conventional two-dimensional cell culture screening systems while simultaneously approaching a level of complexity necessary to consider translation to in vivo animal models. Here, we describe a three-dimensional (3D), in vitro model system to investigate the impact of stromal cell migration from one microenvironment to another at a medium-throughput scale. Importantly, we demonstrate the ability of this workflow to be utilized as a screening tool for collagen-based biomaterial motifs of interest in promoting craniomaxillofacial bone defect repair. Taken together we provide a strategy for interpreting cell-to-cell, cell-to-material, and material-to-material interactions across a multidimensional spatiotemporal scale.

Genetically engineered human induced pluripotent stem cells (hiPSCs) represent a promising platform for regenerative medicine and next-generation immunotherapies. While recent advances enable stroma-free differentiation of hiPSCs into mature CD3⁺TCRαβ⁺ cytotoxic T lymphocytes (CTLs), overall efficiency remains limited. Here, we identify small-molecule modulators that enhance T cell output, particularly at the ProT cell stage. Targeted and stage-specific inhibition of AHR, DOT1L, or GSK3 drives robust maturation from ProT to CD4⁺ immature single-positive (ISP) cells, markedly increasing CD4⁺CD8⁺ populations and augmenting CTL production of up to 2000 fold. hiPSC-derived T (iT) cells matured under these conditions display superior activity in cytotoxicity assays using AMG-701 (BCMAxCD3) or Blinatumomab (CD19xCD3). These effects were reproducible across independent hiPSC lines, diverse hematopoietic progenitor generation methods, and multiple stroma-free differentiation platforms, and were further validated in cord blood CD34⁺ cells. Notably, AHR inhibition enhanced T cell development and promoted B lymphopoiesis, revealing shared regulatory pathways in lymphoid lineage specification. We also demonstrate that the Oct4-activating compound OAC1 functions as a weak AHR inhibitor, partially recapitulating the effects of canonical AHR blockers in both cellular and zebrafish AHR reporter systems. Collectively, our findings define key molecular circuits governing human lymphoid differentiation and establish practical strategies to optimize the yield and function of hiPSC-derived cytotoxic T cells. This work advances the development of both universal and autologous hiPSC-derived T cell therapies, offering a path forward even for patient-specific hiPSC lines with suboptimal T cell differentiation potential.

Effector CD8+ T cells are key cellular drivers of type 1 diabetes (T1D) pathogenesis, yet questions remain regarding the molecular defects leading to altered cytotoxicity, their signature in peripheral tissues, and their receptor specificity. Thus, we analyzed human pancreatic lymph nodes (pLN) using mass cytometry and single cell RNA sequencing (scRNAseq) with combined proteomic and T cell receptor (TCR) profiling. Cytometric analysis revealed an enriched population of T stem-cell memory (TSCM)-like cells (CD8+CD45RA+CD27+CD28+CCR7+CXCR3+ T cells) in T1D pLNs. scRNAseq profiling indicated an elevated inflammatory cytokine gene signature (IFITM3, LTB) along with regulators of terminal differentiation (BCL6, BCL3), coupled with reduced expression of exhaustion-associated genes (DUSP2, NR4A2, TSC22D3) in CD8+ T cells in T1D pLN. Additionally, effector CD8+ T cells expressed features of progenitor exhausted cells (BCL2) in T1D pLN. Immune Response Enrichment Analysis (IREA) indicated IL-15 signaling as a significant driver of these phenotypes. Integrated TCR and transcriptomic analysis revealed a cluster of diverse naïve-like CD8+ T cell clones in T1D pLN. When comparing pLN and pancreatic slice cellular isolates, we observed sharing of effector CD8+ T cells, with upregulation of terminal effector signatures detected within the pancreas relative to paired pLN samples. Multiplex imaging revealed differential localization of TCF1 and TOX expressing T cells in the pancreas, with TCF1+TOX+ cells located in closer proximity to the islets and displaying a mixture of activation and exhaustion-associated phenotypes. Thus, we provide multimodal cellular profiles enriched in T1D tissues for consideration in therapeutic targeting.

Pulmonary fibrosis is a progressive, terminal disease with high mortality. Existing therapeutics are capable of slowing disease progression but are unable to reverse fibrotic lung remodeling, accentuating the importance of studying the mechanisms that underlie lung resilience and repair during fibrosis. Recent literature has suggested that alveolar type 2 (AT2) progenitors undergo transition to stressed Krt8high cells following lung injury. Accumulation of these stressed Krt8high cells has been observed in multiple acute and chronic lung diseases, particularly pulmonary fibrosis. Whether accumulation of Krt8high cells is a direct driver of fibrosis or an epiphenomenon of lung injury remains unclear. We have previously described a genetic model causing transition of AT2 progenitors to a Krt8high cell state following deletion of the lung transcription factor Nkx2-1 specifically in the AT2 progenitor lineage. Here, we use this tractable model of genetic Krt8high cell accumulation to directly evaluate the pathogenic influence of Krt8high cells which are present at the onset of lung fibrosis. Building on recent data, we show that these Nkx2-1-/- Krt8high cells accumulate in a Krt7high/Krt19high/Krt17neg alveolar-basal intermediate state (ABI). Following induction of fibrotic lung injury with inhaled silica, these ABI enter a unique inflammatory state (iABI) that drives severe fibrotic remodeling of the lung via coordination of a fibrotic signaling niche containing inflammatory alveolar fibroblasts (iAF) and pulmonary osteoclast-like cells (POLC). Computational analysis suggests that iABI elaborate pro-inflammatory signals which increase matrix deposition by iAF and drive differentiation of interstitial macrophages to a highly fibrotic POLC-like state. Niche mapping demonstrates that iABI, iAF, and POLCs interact within newly formed fibrotic niches in the lung alveolus, driving widespread fibrosis in animals with pre-existing accumulation of ABI. These data support the conclusion that ABIs actively participate in driving fibrosis after silica-induced lung injury, providing direct evidence that ABI accumulation in fibrotic lung disease is likely pathogenic.

Sodium magnetic resonance imaging ( 23 Na MRI) provides a unique opportunity to probe ionic microenvironments in neural tissue because sodium ions play central roles in membrane electrophysiology, ion transport, and cellular homeostasis. Unlike conventional proton (¹H) MRI, which primarily reflects water distribution and tissue structure, ²³Na MRI is sensitive to ionic compartmentation and quadrupolar interactions arising from the spin-3/2 nature of the sodium nucleus. However, sodium MRI remains technically challenging due to intrinsically low signal sensitivity and rapid biexponential relaxation, particularly when imaging small biological systems. Here, we establish a high-field multinuclear MRI platform for imaging human cerebral organoids at 14 Tesla. Cerebral organoids derived from human induced pluripotent stem cells provide a simplified three-dimensional neural tissue model that enables investigation of ionic microenvironments without vascular or systemic confounds. Using a dual-tuned ¹H/²³Na radiofrequency coil, we performed co-registered structural, diffusion, and sodium imaging of individual fixed organoids. High-resolution ¹H MRI (33-100 μm) revealed pronounced microstructural heterogeneity, while multi-echo ²³Na MRI (300-400 μm) enabled voxel-wise characterization of quadrupolar relaxation behavior. Bi-exponential analysis of the sodium signal decay identified distinct relaxation components (T2* short ≈ 1 ms and T2* long ≈ 12 ms) and revealed spatial heterogeneity in sodium microenvironments across the organoid tissue. These results demonstrate the feasibility of quantitative sodium relaxometry in cortical organoids and establish a multinuclear imaging platform for investigating ionic microenvironment dynamics in three-dimensional neural tissue models.

Conventional bone grafts cannot reliably fulfill the dual requirements of rapid osseoinduction and intrinsic infection-resistance to meet clinical needs. We therefore aimed to overcome this dual challenge by fabricating a novel physiomimetic three-dimensional scaffold. This was achieved by coating the unique nano-grooved cellulosic matrix derived from Cannabis sativa leaf trichomes with reduced graphene oxide (rGO) to mimic the native osteogenic niche. The plant-derived skeleton serves as a ready-made, topographically complex framework, while the rGO coating provides a microenvironment well suited for bone repair. Comprehensive characterization verified a measurable surface energy, hydrophilicity, roughness, and proper conductivity due to rGO coating. Moreover, in vitro examination confirmed that rGO biofunctionalization synergized with the innate nano-topography, dynamically accelerated the osteogenic differentiation of human adipose-derived stem cells. An upregulated expression of key bone markers, COL1A1, RUNX2, and OPN, sustained alkaline phosphatase activity, and augmented deposition of collagen and mineralized matrix exhibited the potential of the proposed approach for efficient osteal regeneration. An equally important finding was the scaffold's inherent antibacterial property against Gram-positive and Gram-negative pathogens. We demonstrated that augmenting a natural cannabis-derived nanostructure with a conductive nanomaterial coating creates a multifaceted therapeutic strategy capable of promoting bone formation and potentially antibacterial effects, addressing two critical obstacles in regenerative orthopedics.

Studying the molecular mechanisms underlying autism spectrum disorders (ASD) requires cellular models capable of capturing cis-regulatory effects and allele-specific gene expression. In this study, we present an approach for generating induced pluripotent stem cells (iPSCs) modified using an adenine base editor (ABE) to introduce synonymous single-nucleotide substitutions in the AUTS2 gene - a candidate involved in ASD pathogenesis. These substitutions serve as allele-specific markers, enabling the tracking of expression differences between normal and rearranged alleles in a cis-regulatory context. We developed a high-efficiency strategy for genotyping clones using amplicon-based next-generation sequencing (NGS). Analysis of over 100 subclones demonstrated that this approach surpasses Sanger sequencing in scalability, sensitivity, and cost-effectiveness. We selected clones with targeted heterozygous substitutions, assessed mosaicism levels, and performed phasing with germline heterozygous variants to confirm monoclonal origin and identify the allele carrying the chromosomal rearrangement. The resulting iPSC lines mark distinct AUTS2 alleles, providing a foundation for analyzing the impact of cis-regulatory elements on gene expression across different cell types. Our findings highlight the practical value of base editors and targeted NGS genotyping in creating cellular models with single-nucleotide substitutions for both basic and applied research.

Acute myeloid leukemia (AML) remains associated with high relapse rates and poor long-term survival, particularly in refractory patients or those ineligible for hematopoietic stem cell transplantation (HSCT). Adoptive transfer of natural killer (NK) cells has emerged as a promising immunotherapeutic strategy due to intrinsic cytotoxicity, low risk of graft-versus-host disease (GVHD), immune restoration capacity, and feasibility of allogeneic off-the-shelf manufacturing.

T cell receptor (TCR) repertoire analysis provides crucial insight into the maturation of the adaptive immune system. In this study, we examined the diversity and structure of TCRβ repertoire in the sorted naïve CD4+, naïve CD8+ and naïve regulatory T cell (Treg) subsets from umbilical cord blood (UCB) at early gestation (24-29 weeks), term (38-39 weeks), and late-born neonates (40-42 weeks), as well as from peripheral blood of children, adults, and older individuals. UCB TCRβ repertoires were characterized by shorter CDR3 regions, increased repertoire convergence, and a higher abundance of public clonotypes, consistent with limited junctional diversity in early development. Our data suggest progressive maturation of the UCB TCRβ repertoire with noticeable changes by late gestation (~29 weeks). Notably, UCB-derived naïve Treg cells displayed distinct TCRβ repertoire features compared with adult Treg cells, indicating subset-specific differences in TCRβ repertoire shaping. Across age groups, we observed an age-dependent shift in TCRβ repertoire structure associated with changes in TRBV gene usage with a progressive decrease in the predicted binding strength of CDR2, particularly in repertoires of naïve Treg and naïve CD8+; T cells. Together, these results provide insights into TCRβ repertoire formation during human ontogeny and highlight subset-specific trajectories during early life.

Tumor heterogeneity is a fundamental driver of therapeutic resistance across solid malignancies, arising from genetic, epigenetic, phenotypic, spatial, temporal, and microenvironmental diversity. In tumors developing at mucosal barrier sites, these heterogeneous features are further shaped by the unique immunological context of mucosal tissues, where immune tolerance, chronic inflammation, and continuous antigen exposure create permissive environments for immune escape and adaptive resistance. Accumulating evidence indicates that myeloid cell plasticity, including functional diversification of granulocytes, macrophages, monocytes, and dendritic cells, represents a critical interface between tumor-intrinsic heterogeneity and mucosal immune regulation. These myeloid populations contribute to spatially organized immunosuppressive niches, altered antigen processing and presentation, and therapy-induced immune remodeling, collectively influencing responses to chemotherapy, targeted therapy, and immunotherapy. Advances in single-cell sequencing, spatial transcriptomics, multiplex imaging, and liquid biopsy technologies, coupled with artificial intelligence-enabled analytics, have enabled high-resolution mapping of heterogeneous tumor immune landscapes and revealed convergent resistance mechanisms driven by clonal selection, phenotypic plasticity, microenvironmental buffering, and myeloid-mediated immune suppression. In this review, we synthesize mechanistic and clinical evidence across major cancer types, including colorectal and lung cancers as archetypal mucosal tumors, along with broader examples from breast cancer, melanoma, and immunotherapy-treated malignancies. We highlight how heterogeneous cellular states and immune niches influence clinical outcomes. Finally, we discuss emerging translational strategies to overcome resistance, including rational combination regimens, epigenetic and metabolic targeting, adaptive therapy, myeloid reprogramming approaches, and real-time biomarker monitoring. These approaches aim to restore effective anti-tumor immunity while accounting for the unique constraints of mucosal barrier tissue.