Biology subjects

Huynh, N. P.

Publications and source records attributed to Huynh, N. P..

5 recordsLinked to original sources

bioRxiv2026

Generation and validation of a human iPSC-derived TDP-43 knockout model for ALS disease modeling.

Nuclear depletion and cytoplasmic aggregation of TDP-43 occur in [~]97% of amyotrophic lateral sclerosis (ALS) cases and disrupt RNA processing through aberrant cryptic exon inclusion. Existing cellular models rely on partial knockdown, TARDBP mutations, or pharmacological stress, each with limitations. Here, we generated homozygous TARDBP-knockout human iPSC lines using CRISPR-Cas9 genome editing and differentiated them into spinal motor neurons (MNs). Knockout MNs demonstrated [~]16-fold lower differentiation efficiency than isogenic controls but retained neuronal marker expression. TDP-43 loss induced widespread cryptic exon inclusion and depletion of STMN2, UNC13A, and G3BP1. Integration of the CUTS splice biosensor yielded up to 4.5-fold cryptic GFP induction in knockout MNs, providing a reporter-based readout of TDP-43 dysfunction. Further, we validated the cardiac glycosides digoxin and ouabain as modulators of bortezomib-induced TDP-43 pathology. This genetically defined iPSC-derived MN model provides a platform for mechanistic and therapeutic interrogation of TDP-43-driven neurodegeneration in ALS.

neuroscience↗

bioRxiv2025

Histone hyperacetylation-linked upregulation of KRAB zinc finger proteins impedes glial differentiation in Huntington's disease

Glial differentiation is impaired in Huntington disease (HD), contributing to both the synaptic dysfunction and hypomyelination of HD. Through combined epigenomic and transcriptomic profiling, we found that glial progenitor cells (hGPCs) generated from HD-derived human embryonic stem cells exhibit persistent histone hyperacetylation, enabling the ectopic expression of a broad set of KRAB zinc finger protein (KZFP) transcriptional repressors. Single-cell RNA-Seq analysis of HD hGPCs revealed that their aberrant KZFP expression was attended by the persistent expression of neural progenitor-stage genes relative to wild-type hGPCs. The HD hGPCs over-expressed the MYST family histone acetyltransferase KAT6B, which led to their hyperacetylation at H3K9 and associated DNA demethylation, and displayed abnormally open chromatin, particularly at promoters of chromosome 19 KZFP gene clusters. Among those KZFPs most differentially activated in HD hGPCs was the primate-specific ZNF98, whose overexpression in wild-type hGPCs recapitulated the HD-associated suppression of glial development. These data implicate abnormal histone hyperacetylation in HD glial progenitor cells, and its associated over-expression of recently evolved KZFP transcriptional repressors, as a critical mechanism by which both astrocytic and oligodendrocytic differentiation are impaired in HD.

neuroscience↗

bioRxiv2019

Single Cell RNA Sequencing Reveals Heterogeneity of Human MSC Chondrogenesis: Lasso Regularized Logistic Regression to Identify Gene and Regulatory Signatures

Bone marrow-derived mesenchymal stem cells (MSCs) exhibit the potential to undergo chondrogenesis in vitro, forming de novo tissues with a cartilage-like extracellular matrix that is rich in glycosaminoglycan and collagen type II. However, it is now apparent that MSCs comprise an inhomogeneous population of cells, and the fate of individual subpopulations during this differentiation process is not well understood. We analyzed the trajectory of MSC differentiation during chondrogenesis using single cell RNA sequencing (scRNA-seq). Using a machine learning technique - lasso regularized logistic regression - we showed that multiple subpopulations of cells existed at all stages during MSC chondrogenesis and were better-defined by transcription factor activity rather than gene expression. Trajectory analysis indicated that subpopulations of MSCs were not intrinsically specified or restricted, but instead remained multipotent and could differentiate into three main cell types: cartilage, hypertrophic cartilage, and bone. Lasso regularized logistic regression showed several advances in scRNA-seq analysis, namely identification of a small number of highly influential genes or transcription factors for downstream validation, and cell type classification with high accuracy. Additionally, we showed that MSC differentiation trajectory may exhibit donor to donor variation, although key influential pathways were comparable between donors. Our data provide an important resource to study gene expression and to deconstruct gene regulatory networks in MSC differentiation.

genomics↗

bioRxiv2019

Single cell RNA-sequencing reveals cellular heterogeneity and trajectories of lineage specification during murine embryonic limb development

The coordinated spatial and temporal regulation of gene expression in the murine hindlimb determines the identity of mesenchymal progenitors and the development of diversity of musculoskeletal tissues they form. Hindlimb development has historically been studied with lineage tracing of individual genes selected a priori, or at the bulk tissue level, which does not allow for the determination of single cell transcriptional programs yielding mature cell types and tissues. To identify the cellular trajectories of lineage specification during limb bud development, we used single cell mRNA sequencing (scRNA-seq) to profile the developing murine hindlimb between embryonic days (E)11.5-E18.5. We found cell type heterogeneity at all time points, and the expected cell types that form the mouse hindlimb. In addition, we used RNA fluorescence in situ hybridization (FISH) to examine the spatial locations of cell types and cell trajectories to understand the ancestral continuum of cell maturation. This data provides a resource for the transcriptional program of hindlimb development that will support future studies of musculoskeletal development and generate hypotheses for tissue regeneration.

molecular biology↗

bioRxiv2019

Long non-coding RNA GRASLND enhances chondrogenesis via suppression of interferon type II signaling pathway

Long non-coding RNAs (lncRNAs) play critical roles in regulating gene expression and cellular processes; however, their roles in musculoskeletal development, disease, and regeneration remain poorly understood. Here, we identified a novel lncRNA, Glycosaminoglycan Regulatory ASsociated Long Non-coDing RNA (GRASLND) as a regulator of mesenchymal stem cell (MSC) chondrogenesis, and we investigated its basic molecular mechanism and its potential application towards regenerative medicine. GRASLND, a primate-specific lncRNA, is upregulated during MSC chondrogenesis and appears to act directly downstream of SRY-Box 9 (SOX9), but not Transforming Growth Factor Beta 3 (TGF-{beta}3). Utilizing the established model of pellet formation for MSC chondrogenesis, we showed that the silencing of GRASLND resulted in lower accumulation of cartilage-like extracellular matrix, while GRASLND overexpression, either via transgene ectopic expression or by endogenous activation via CRISPR, significantly enhanced cartilage matrix production. GRASLND acts to inhibit interferon gamma (IFN-{gamma}) by binding to Eukaryotic Initiation Factor-2 Kinase EIF2AK2. We further demonstrated that GRASLND exhibits a protective effect in engineered cartilage against interferon type II across different sources of chondroprogenitor cells. Our results indicate an important role of GRASLND in regulating stem cell chondrogenesis, as well as its therapeutic potential in the treatment of cartilage-related diseases, such as osteoarthritis.\n\nSignificanceLong non-coding RNAs (lncRNAs) play critical roles in gene regulation and cellular physiology; however, the role of lncRNAs in controlling stem cell chondrogenesis remains to be determined. Here, we utilized next generation sequencing of adult stem cell chondrogenesis to identify a set of potential lncRNA candidates involved in this process. We identified lncRNA Glycosaminoglycan Regulatory ASsociated Long Non-coDing RNA (GRASLND) and characterized its molecular mechanism of action. We described a novel role of GRASLND in positive regulation of chondrogenesis via its inhibition of type II interferon. Importantly, we showed that overexpression of GRASLND augments stem cell chondrogenesis, providing a promising approach to enhancing stem cell chondrogenesis and cartilage regeneration.

cell biology↗