Biology subjects

Kim, H. H.

Publications and source records attributed to Kim, H. H..

17 recordsLinked to original sources

bioRxiv2026

Lineage tracing reveals asynchronous fate restriction in mouse embryos and stem cell-derived embryo models

How a flexible mammalian embryo gives rise to a reproducible body plan remains unclear, partly because cell lineages cannot be followed continuously in vivo. We combined DNA Typewriter lineage recording with tetraploid complementation to reconstruct high-resolution single-cell genealogies across mouse development and built FateVec, an analysis framework that infers fate-bias dynamics and lineage-associated transcriptional programs from reconstructed phylogenies. Loss of developmental potential proved progressive and asynchronous, with multiple lineages acquiring detectable fate bias before morphological diversification at gastrulation. Within the neural crest, most cells remained multipotent over an extended window, while independent founder clades differed markedly in restriction timing. Extending lineage recording to a stem-cell-derived embryo model revealed that cells with shared transcriptional identities can arise from distinct lineage relationships. We further found embryonic-lineage contributions to extraembryonic-like states, exposing an unexplored lineage plasticity. This lineage-resolved view of embryogenesis reveals that cell identity alone predicts neither developmental ancestry nor the timing of fate restriction.

developmental biology↗

bioRxiv2026

Time-resolved lineage recording reveals a pre-existing, heritable cell state underlying metastatic potential

Metastasis causes most cancer deaths1,2, yet no recurrent mutation specifically drives it3,4, raising the possibility that metastatic potential is a non-genetic yet heritable cell state. Classic experiments established that metastatically predisposed subclones pre-exist within a tumor and that these predispositions are inherited over many cell divisions5, but what molecular states or factors underlie this predisposition remain unknown. While previous lineage recording studies6,7 mapped how tumors disseminate, their recording sites saturate too quickly to resolve when a lineage branched, or to attribute a state to its founder. Here we show, using a DNA Typewriter lineage recorder8 with nearly 1,000 recording sites in lung cancer cells, that metastatic potential is already present before dissemination, with colonization predicted by a pre-existing glycolytic state and further spread by expression of ENO1, a glycolytic enzyme that also moonlights as a cell-surface plasminogen receptor9. Profiling the pre-transplant cells and the post-transplantation tumors for both their transcriptomes and their lineage recordings, we reconstructed time-resolved lineage trees across three orthotopically transplanted mice. These trees trace each liver metastasis to a single founder of known pre-transplant state, dating each dissemination event from the primary lung. When every clone was scored before transplant against 349 genes recurrently heritable in vitro, both that set and the glycolytic state independently shifted a clones odds of colonizing the lung. At the gene level, sixteen genes were both heritable and predictive of colonization, and ENO1 alone also predicted which established clones spread further. Hypoxia, the program most strongly associated with phylogenetic fitness within the metastases, did not predict colonization when scored before transplant, separating niche-selected traits from the inherited cell state. Metastatic potential in this system is therefore transmitted along the lineage rather than acquired after seeding. Looking forward, we anticipate that time-resolved lineage recorders will enable the separation of the heritable and acquired components of the cellular heterogeneity seen in single-cell studies of tumor progression and drug tolerance.

cancer biology↗

bioRxiv2026

Developmental plasticity of cancer-associated fibroblasts contributes to the malignant progression of gastric cancers

Cancer-associated fibroblasts are transcriptionally heterogeneous, yet how their reprogramming shapes gastric cancer progression remains unclear. Integrating single-cell and spatial transcriptomics, patient-derived fibroblast co-culture, and clinical cohorts, we identified a spatially organized continuum of extracellular matrix-producing fibroblasts. An inferred trajectory connected homeostatic ECMh through inflammatory-chemoattractive ECMi to tissue-remodeling ECMr, with increasing GLIS2 regulon activity and ECMr enrichment near the tumor-stroma interface. ECMr co-culture induced a non-EMT epithelial program characterized by coordinated laminin-332 subunit expression, hemidesmosome-associated genes, a matrix-remodeling protease profile, and reduced proliferative activity. Longitudinal imaging showed progressive cancer-cell elongation and positional displacement over the fibroblast layer. ECMr-associated GLIS2 regulon activity in resected primary tumors was associated with shorter peritoneal metastasis-free survival across two gastric cancer cohorts, with a similar association in colorectal cancer. A peritoneal metastasis recapitulated the corresponding stromal and epithelial organization. Together, these findings define an ECMr-centered stromal-epithelial wound-repair circuit linked to subsequent peritoneal dissemination.

cancer biology↗

bioRxiv2026

Reprogramming Dedifferentiation Regulatory Networks Preserves Human Chondrocyte Phenotypes

Chondrocyte-based cartilage repair strategies such as autologous chondrocyte implantation (ACI) require extensive in vitro expansion to obtain clinically relevant cell numbers. However, this expansion step progressively drives chondrocyte dedifferentiation, reducing matrix-forming capacity and contributing to variable repair outcomes. To better understand this process, we used single-nucleus multiome profiling (snRNA-Seq + snATAC-Seq) to define the transcriptional and chromatin accessibility programs underlying human chondrocyte dedifferentiation during expansion. Multiome integration across passages revealed a continuous dedifferentiation trajectory accompanied by coordinated remodeling of gene expression and chromatin accessibility, identifying chromatin destabilization as an early regulatory event during phenotype loss. Guided by these regulatory signatures, we screened available small-molecule inhibitors targeting candidate pathways and found that Fludarabine most consistently preserved chondrocyte identity during early expansion. Fludarabine was associated with suppression of STAT1-related programs and early stabilization of the chromatin landscape prior to broader transcriptional recovery. Functionally, treated cells demonstrated enhanced matrix-forming capacity in chondrogenic pellet culture and significantly increased nascent protein synthesis in 3D hydrogel culture, with biosynthetic output approaching unexpanded controls by day 21. Together, these findings identify chromatin stability as a key regulatory determinant of expansion-associated chondrocyte dedifferentiation and establish a pharmacologic strategy to preserve chondrocyte functional potency during cell manufacturing for cartilage repair.

bioengineering↗

bioRxiv2026

Reverse electron transfer at mitochondrial complex I restrains dopaminergic neuron activity to promote early-life sleep in Drosophila

Sleep is especially abundant during early life, yet the cellular mechanisms that maintain elevated juvenile sleep drive remain poorly understood. To identify cell-intrinsic regulators of juvenile sleep, we profiled gene expression in Drosophila dopaminergic neurons (DANs) at different ages and performed a targeted RNAi screen of genes with enriched juvenile expression. We found that the magnitude of mitochondrial complex I (MCI) disruption produced strikingly distinct physiological outcomes. Severe MCI loss-of-function caused profound mitochondrial dysfunction, reduced dopaminergic activity, and locomotor impairment. In contrast, partial MCI inhibition left overall mitochondrial function intact but instead reduced sleep, with disproportionately strong effects on juvenile sleep fragmentation and depth, consistent with a gain of dopaminergic signaling. Multiple genetic manipulations that deplete the reduced coenzyme Q pool (CoQH2) converged on a phenotype of increased dopaminergic activity and wakefulness, indicating that coenzyme Q redox balance itself regulates dopaminergic output. These findings support a model in which CoQH2-driven reverse electron transfer (RET) at MCI restrains dopaminergic arousal. By dissociating mitochondrial redox signaling from catastrophic mitochondrial failure, our work identifies a physiological role for coenzyme Q-dependent signaling in the regulation of neural activity and behavioral state. More broadly, these findings suggest that sleep phenotypes may serve as sensitive indicators of emerging mitochondrial dysfunction and reveal a potential link between the regulation of early-life sleep and later dopaminergic vulnerability.

neuroscience↗

bioRxiv2026

The effects of elevated seawater pH and total alkalinity following dosing of sodium hydroxide in Calanus finmarchicus

Ocean Alkalinity Enhancement (OAE) is a marine carbon dioxide removal (mCDR) strategy that involves adding alkaline substances to surface waters to enhance CO2 uptake and storage. The dispersal of alkaline materials such as sodium hydroxide (NaOH) into seawater can cause rapid increases in pH and total alkalinity (TA) that substantially exceeds natural variability in marine environments. Such fluctuations may negatively affect marine life, especially small animals like copepods who cannot avoid OAE plumes and whose physiological processes could be disrupted by large and rapid shifts in seawater pH. To address knowledge gaps regarding potential biological impacts of OAE, we studied these effects in Calanus finmarchicus, a keystone copepod species in the Northwest Atlantic Ocean. We exposed C. finmarchicus from the late juvenile copepodite stages and adult females to NaOH-dosed seawater at pH 10.5 ([~]5,000 {micro}mol kg-1 TA) and pH 9.0 ([~]3,150 {micro}mol kg-1 TA) for durations that reflect expected short-term exposure times during field OAE deployments (pH 10.5: 1, 5, 10 minutes; pH 9.0: 1, 15, 30 minutes). None of the treatment combinations resulted in mortality immediately after the initial exposure. Individuals were monitored for survival for 72 hours post-exposure (hpe), and only one treatment group (juveniles exposed to pH 10.5 for 10 minutes) showed a significant reduction in final survival; no other pH-duration combination showed increased mortality. Effects on the ability to initiate an escape response were more substantial. Adult females treated with pH 10.5 for 5 or 10 minutes showed a significant reduction in escape response immediately after exposure. In contrast, juveniles showed no immediate change in escape response following exposure to pH 10.5 or pH 9.0, although juveniles exposed to pH 10.5 for 10 minutes exhibited reduced escape response at 24 hpe. Using microrespirometry, we measured oxygen consumption following a 10-minute exposure to pH 10.5 and detected no effect on routine metabolic rate immediately post-exposure or at 12 hpe. Overall, our results suggest that C. finmarchicus is relatively tolerant to short-term exposures to very high pH and alkalinity. Future work should prioritize longer-term exposure under more moderate ocean OAE conditions.

ecology↗

bioRxiv2025

From microbial diversity to function; evaluating dimensionality reduction methods

Artificial Intelligence (AI), and more specifically Machine Learning (ML), have become an increasingly prevalent tool in microbial oceanography. The high dimensionality of microbial diversity data from omics observations is highly suitable for ML analysis, with many recent studies showcasing their utility for exploratory ecological feature finding and process prediction. Here, we apply three well-documented dimensionality reduction methods including Principal Coordinate Analysis (PCoA), Self Organizing Maps (SOM), and Weighted Gene Correlation Network Analysis (WGCNA), to near daily 16S rRNA gene amplicon sequencing data from the 2019-2020 MOSAiC International Arctic Drift Expedition. We compare the k-means clustering outputs from these methods to extract functionally distinct seasonal microbial ecotypes in the surface Arctic Ocean. Our results indicate the SOM method outperforms a more traditional PCoA ordination, identifying a greater number of metabolically distinct functional groups. We then investigate the importance of including biological context in dimensionality reduction by comparing functional outputs to a taxa clustering approach using a k-means adapted WGCNA correlation network. Regardless of data input, all 3 methods identified 3-4 recurrent ecotypes with distinct taxonomic and functional cut-offs driven by seasonality, water mass, and substrate turnover. Ultimately, these results reinforce such methodologies as a meaningful translator in the mining of historical amplicon datasets to address modern mechanistic questions and incorporate greater ecotype diversity into mechanistic biogeochemical models. ImportanceConnecting microbial community structure to ecosystem function is an important step in accurately modeling climate-relevant biogeochemical processes yet remains a major challenge in microbial oceanography. This manuscript demonstrates how emerging machine learning approaches can establish this connection by uncovering recurrent ecological patterns in Arctic Ocean microbial communities. Using near-daily 16S rRNA gene and supplementary metagenome data from the MOSAiC drift expedition, we identified distinct "ecotypes," or groups of microbes that perform differentiable functional roles within the ecosystem. Importantly, our methods reveal new connections between microbial identity and function that traditional analyses may overlook. It is possible such techniques could be applied to historical amplicon datasets, allowing scientists to revisit and reinterpret existing data to better understand how polar ecosystems are responding to environmental change and to improve future predictive climate models.

ecology↗

bioRxiv2025

O-SNAP: A comprehensive pipeline for spatial profiling of chromatin architecture

We present O-SNAP (Objective Single-Molecule Nuclear Architecture Profiler), a comprehensive pipeline for the automated extraction, comparison, and classification of nuclear features from single-molecule localization microscopy (SMLM) data. O-SNAP quantifies 144 interpretable, biologically grounded spatial features describing chromatin organization or histone mark distributions at nanoscale resolution. The pipeline includes modules for pairwise comparison of features using volcano plots, feature set enrichment analysis, robust feature selection and classification of cell states, and pseudotime trajectory inference. We validate O-SNAP across diverse biological contexts, including fibroblast-to-stem cell reprogramming, tendon disease, histone variant sensitivity to oxidative stress, and chondrocyte de-differentiation, demonstrating its ability to detect subtle changes in nanoscale chromatin organization across diverse biological transitions.

biophysics↗

bioRxiv2023

Saturation resistance profiling of EGFR variants against tyrosine kinase inhibitors using prime editing

Variants of uncertain significance (VUS) hamper the clinical application of genetic information. For example, in treating lung cancer with tyrosine kinase inhibitors (TKIs), many epidermal growth factor receptor (EGFR) variants remain classified as VUS with respect to TKI sensitivity1,2. Such incomplete resistance profiles hinder clinicians from selecting optimal therapeutic agents3,4. A high-throughput approach that can evaluate the functional effects of single nucleotide variants (SNVs) could reduce the number of VUS. Here we introduce SynPrime, a method based on prime editing that enabled the generation and functional evaluation of 2,476 SNVs in the EGFR gene, including 99% of all possible variants in the canonical tyrosine kinase domain (exons 18 to 21). We determined resistance profiles of 95% (= 1,726/1,817) of all possible EGFR protein variants encoded in the whole tyrosine kinase domain (exons 18 to 24) against afatinib, osimertinib, and osimertinib in the presence of the co-occurring mutation T790M, in PC-9 cells. SynPrime, which uses direct sequencing of endogenous regions to identify SNVs, provided more accurate functional evaluations than a guide RNA abundance-based approach. Our study has the potential to substantially improve the precision of therapeutic choices in clinical settings and contribute to addressing the issue of VUS by being applied to other genes.

cancer biology↗

bioRxiv2022

Sniper2L, a high-fidelity Cas9 variant with high activity

Although several high-fidelity SpCas9 variants that have reduced activities at mismatched target sequences have been reported, it has been observed that this increased specificity is associated with reduced on-target activity, limiting the applications of the high-fidelity variants when efficient genome editing is required. Here, we developed an improved version of Sniper-Cas9, Sniper2L, which represents an exception to this trade-off trend as it showed higher specificity with retained high activity. We evaluated Sniper2L activities at a large number of target sequences, and developed DeepSniper, a deep-learning model that can predict the activity of Sniper2L. We also confirmed that Sniper2L can induce highly efficient and specific editing at a large number of target sequences when it is delivered as a ribonucleoprotein complex. Mechanically, the high specificity of Sniper2L originates from its superior ability to avoid unwinding a target DNA containing even a single mismatch. We envision that Sniper2L will be useful when efficient and specific genome editing is required.

bioengineering↗

bioRxiv2022

Disease-causing variant recommendation system for clinical genome interpretation with adjusted scores for artefactual variants

BackgroundIn the process of finding the causative variant of rare diseases (RD), accurate assessment and prioritization of genetic variants is essential. Although quality control (QC) of genetic variants is strictly performed, the presence of artefactual variants in the remaining set of variants can deteriorate the process. Variant QC and prioritization have been treated as separate processes, leading to limited efficiency and risk of misdiagnosis. ResultsWe developed a disease-causing variant recommendation system that integrates quality control into variant prioritization by adjusting scores for artefactual variants. We confirmed that the QC-related features of the variants contribute to a significant performance improvement. For genomic data from 2,878 patients with rare disorders, the recall rate of finding causative variants was 0.961 for the top 5 ranked variants. We also found that our system recognized the anomaly of QC-related features, so that the scores of artifactual variants to be disease-causing were assessed relatively low. ConclusionsIntegration of variant QC and prioritization help reduce the risk of misdiagnosis based on artefactual variants and increase the effectiveness of clinical genome interpretation.

genetics↗

bioRxiv2022

Enhanced mTORC1 signaling and Protein Synthesis in Parkinson Disease Pathogenesis

Pathologic -syn destabilizes the TSC 1 and 2 complex leading to mTORC1 activation, enhanced protein translation and neurodegeneration in PD. AbstractPathological -synuclein (-syn) plays an important role in the pathogenesis of -synucleinopathies such as Parkinsons disease (PD). Disruption of protein homeostasis is thought be central to PD pathogenesis, however the molecular mechanism of this deregulation is poorly understood. Here we report that pathologic -syn binds to tuberous sclerosis protein (TSC) 2 and destabilizes the TSC1-TSC2 complex leading to activation of the mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) and enhanced mRNA translation. Dopamine neuron loss, behavioral deficits and aberrant biochemical signaling in the -syn preformed fibril (PFF) and Drosophila -syn transgenic models of pathologic -syn induced degeneration were attenuated by genetic and pharmacologic inhibition of mTOR and protein translation. Our findings establish a potential molecular mechanism by which pathologic -syn activates mTORC1 leading to enhanced protein translation and concomitant neurodegeneration in PD.

neuroscience↗

bioRxiv2021

A merger between compatible but divergent genomes supports allopolyploidization in the Brassicaceae family

Hybridization and polyploidization are pivotal to plant evolution. Genetic crosses between distantly related species rarely occur in nature mainly due to reproductive barriers but how such hurdles can be overcome is largely unknown. xBrassicoraphanus is a fertile intergeneric allopolyploid synthesized between Brassica rapa and Raphanus sativus in the Brassicaceae family. Genomes of B. rapa and R. sativus are diverged enough to suppress synapsis formation between non-homologous progenitor chromosomes during meiosis, and we found that both genomes reside in the single nucleus of xBrassicoraphanus without genome loss or rearrangement. Expressions of syntenic orthologs identified in B. rapa and R. sativus were adjusted to a hybrid nuclear environment of xBrassicoraphanus, which necessitates reconfiguration of transcription network by rewiring cis-trans interactions. B. rapa coding sequences have a higher level of gene-body methylation than R. sativus, and such methylation asymmetry is maintained in xBrassicoraphanus. B. rapa-originated transposable elements were transcriptionally silenced in xBrassicoraphanus, rendered by gain of CHG methylation in trans via small RNAs derived from the same sequences of R. sativus subgenome. Our work proposes that not only transcription compatibility but also a certain extent of genome divergence supports hybrid genome stabilization, which may explain great diversification and expansion of angiosperms during evolution.

plant biology↗

bioRxiv2021

Prime editing enables precise genome editing in mouse liver and retina

Prime editing can induce any small-sized genetic change without donor DNA or double strand breaks. However, it has not been investigated whether prime editing is possible in postnatal animals. Here we delivered prime editors 2 and 3 into a mouse model of hereditary tyrosinemia, a genetic liver disease, using hydrodynamic injection, which corrected the disease-causing mutation and rescued the phenotype. We also achieved prime editing in the retina and retina pigment epithelium in wild-type mice by delivering prime editor 3 using trans-splicing adeno-associated virus. Deep sequencing showed that unintended edits at or near the target site or off-target effects were not detectable except for low levels (0% to 1.2%) of indels when PE3, but not PE2, was used. Our study suggests that precise, prime editor-mediated genome editing is possible in somatic cells of adult animals.

bioengineering↗

bioRxiv2020

Successful Correction of ALD Patient-derived iPSCs Using CRISPR/Cas9

X-linked adrenoleukodystrophy (ALD) caused by the ABCD1 mutation, is the most common inherited peroxisomal disease. It is characterized by three phenotypes: inflammatory cerebral demyelination, progressive myelopathy, and adrenal insufficiency, but there is no genotype-phenotype correlation. Hematopoietic stem cell transplantation can only be used in a few patients in the early phase of cerebral inflammation; therefore, most affected patients have no curative option. Previously, we reported the generation of an ALD patient-derived iPSC model and its differentiation to oligodendrocytes. In this study, we have performed the first genome editing of ALD patient-derived iPSCs using homology-directed repair (HDR). The mutation site, c.1534G>A [GenBank: NM_000033.4], was corrected by introducing ssODN and the CRISPR/Cas9 system. The cell line exhibited normal ALD protein expression following genome editing. We differentiated the intermediate oligodendrocytes from mutation-corrected iPSCs and the metabolic derangement of ALD tended to correct but was not statistically significant. Mutation-corrected iPSCs from ALD patient can be used in research into the pathophysiology of and therapeutics for ALD.

neuroscience↗

bioRxiv2019

Scarless Enriched selection of Genome edited Human Pluripotent Stem Cells Using Induced Drug Resistance

An efficient gene editing technique for use in human pluripotent stem cells (hPSCs) would have great potential value in regenerative medicine, as well as in drug discovery based on isogenic human disease models. However, the extremely low efficiency of gene editing in hPSCs is a major technical hurdle that remains to be resolved. Previously, we demonstrated that YM155, a survivin inhibitor developed as an anti-cancer drug, induces highly selective cell death in undifferentiated hPSCs. In this study, we demonstrated that the high cytotoxicity of YM155 in hPSCs, which is mediated by selective cellular uptake of the drug, is due to high expression of SLC35F2 in these cells. Consistent with this, knockout of SLC35F2 with CRISPR-Cas9 or depletion with siRNAs made hPSCs highly resistant to YM155. Simultaneous gene editing of a gene of interest and transient knockdown of SLC35F2 following YM155 treatment enabled genome-edited hPSCs to survive because YM155 resistance was temporarily induced, thereby achieving enriched selection of genome-edited clonal populations. This precise and efficient genome editing approach took as little as 3 weeks without cell sorting or introduction of additional genes.

developmental biology↗

bioRxiv2018

Somatic uniparental disomy with a rare EFL1 variant causes Shwachman-Diamond syndrome through dysregulating ribosomal protein synthesis

Shwachman-Diamond syndrome (SDS; OMIM: #260400) is caused by variants in SBDS (Shwachman-Bodian-Diamond syndrome gene), which encodes a protein that plays an important role in ribosome assembly. Recent reports suggest that recessive variants in EFL1 are also responsible for SDS. However, the precise genetic mechanism that leads to EFL1-induced SDS remains incompletely understood. Here we present three unrelated Korean SDS patients that carry biallelic pathogenic variants in EFL1 with biased allele frequencies, resulting from a bone marrow-specific somatic uniparental disomy (UPD) in chromosome 15. The recombination events generated cells that were homozygous for the relatively milder variant, allowing for the evasion of catastrophic physiological consequences. Still, the milder EFL1 variant was solely able to impair 80S ribosome assembly and induce SDS features in cell line, zebrafish, and mouse models. The loss of EFL1 resulted in a pronounced inhibition of terminal oligo-pyrimidine element-containing ribosomal protein transcript 80S assembly. Therefore, we propose a more accurate pathogenesis mechanism of EFL1 dysfunction that eventually leads to aberrant translational control and ribosomopathy.

genetics↗