Project description:Purpose: Corneal endothelial dysfunction leads to irreversible blindness, yet treatment remains constrained by a global shortage of donor tissue. This study aimed to develop a donor-independent strategy for generating functional corneal endothelial cells (CECs) by reprogramming murine neural crest cells (NCCs) through pharmacological inhibition of TGF-β signaling. Methods: NCCs were genetically labeled and isolated from E13.5 Wnt1-Cre;Rosa26-LSL-tdTomato mouse embryos by fluorescence-activated cell sorting. Sorted NCCs were treated with the selective TGF-β inhibitor SB431542 to induce conversion into induced corneal endothelial–like cells (iCECs). Cell identity was validated by immunofluorescence, flow cytometry, wound-healing and endocytic uptake assays, and bulk RNA sequencing with comparison to primary CECs and NCCs. Therapeutic efficacy was assessed by transplanting iCECs into a rabbit model of corneal endothelial dysfunction induced by descemetorhexis. Results: SB431542 treatment efficiently converted NCCs into polygonal, cobblestone-like iCECs that expressed canonical CEC markers (ZO-1, Na⁺/K⁺-ATPase, AQP1, Laminin) while silencing neural crest genes. Approximately 85% of iCECs were Ki67-positive, and cells achieved complete wound closure within 24 hours. Transcriptomic profiling confirmed that iCECs closely resembled primary CECs and were clearly distinct from NCCs. Following transplantation, iCECs reconstituted a confluent endothelial monolayer, normalized central corneal thickness, and restored corneal transparency by Day 35, whereas untreated controls remained opaque. Conclusions: Targeted TGF-β inhibition enables efficient lineage conversion of NCCs into functional corneal endothelial–like cells with robust proliferative capacity and in vivo therapeutic efficacy. This pharmacologically defined strategy provides a scalable, donor-independent cell source and offers a promising therapeutic approach for corneal endothelial diseases.
Project description:Purpose: The goal of this study was to evaluate changes in the transcriptome profile during periocular neural crest differentiation into corneal endothelium and keratocytes. Methods: RNA profiles of chick embryonic day (E3) periocular neural crest, E5 corneal endothelium, and E7 corneal endothelium plus keratocytes were generated in triplicate by deep sequencing using Illumina HS4000. Bowtie2 and HISAT were used to map clean reads to reference gene and genome, respectively. An average mapping ratio of 76.26% to the reference gene and 93.10% to the genome were generated with Galgal5 reference assembly.Transcripts were normalized and presented as Fragments Per Kilobase Million (FPKM). Differentially expressed genes between pNCvsEn and pNCvsKEn were examined. Expression of some candidate genes was validated by in situ hybridization. Results: 790 transcripts were enriched between pNC and En, and 865 transcripts were enriched between pNC and KEn. Enriched transcripts correspond with KEGG pathways involved in cell proliferation, synthesis of extracellular matrix, focal adhesion, metabolism, and cancer. The RNA-Seq data serves as platform for further analyses of the molecular networks involved in NCC differentiation into corneal cells, and provides insights into genes involved in corneal dysgenesis and adult diseases.
Project description:We utilized quantitative analyses of the proteome, transcriptome, and ubiquitinome to study how ubiquitination and NEDD4 control neural crest cell survival and stem-cell-like properties. We report 276 novel NEDD4 targets in neural crest cells and show that loss of NEDD4 leads to a striking global reduction in specific ubiquitin lysine linkages.
Project description:Cells were isolated from mouse embryonic neural crest stem cells at culture day 2 (NCSC), from day 7 in vitro differentiated progeny (NCP) and day 2 epidermal neural crest stem cells from bulge explants of adult whisker follicles (EPI-NCSC). Keywords: LongSAGE embryonic neural crest stem cells at culture day 2 (NCSC), from day 7 in vitro differentiated progeny (NCP) and day 2 epidermal neural crest stem cells from bulge explants of adult whisker follicles (EPI-NCSC).
Project description:Neural crest (NC) cells contribute to the development of many complex tissues. The abnormal development of NC cells accounts for a number of congenital birth defects. Generating NC cells, and more specifically NC subpopulations such as cranial, cardiac, and trunk NC cells from human induced pluripotent stem (iPS) cells and human embryonic stem (ES) cells presents a valuable tool to model and study human NC development and disease. Here we provide a robust, efficient, and reproducible protocol for the differentiation of human iPS and ES cells into NC cells. The protocol has been validated in multiple human pluripotent stem cell lines and yields relatively pure NC cell populations in eight days. The resulting cells can be propagated and retain NC marker expression over multiple passages. The NC cells show proper cell specification and can develop into NC-derived cell lineages including smooth muscle cells, peripheral neurons, and Schwann cells. Additionally, the NC cells are functional and migrate to appropriate chemoattractants such as SDF-1, Fgf8b, BMP2, and Wnt3a. Importantly, this method generates all NC subpopulations (cranial, cardiac, and trunk) providing a great advantage to readily available NC differentiation methods. Neural crest cells derived from human induced pluripotent stem cells were profiled using Affymetrix Gene 1.0 arrays to identify differential gene expression changes and alternative exons from the open-source software AltAnalyze. An FDR adjusted emperical Bayes moderated t-test p < 0.05 was used to identify differentially expressed Ensembl genes and GO-Elite used to identify biologically relevant, Ontology, pathway and gene-set categories. Alternative exons were obtained using the FIRMA analysis option and default thresholds. Other array neural crest array and RNA-Seq dataset were compared to this to identify common and distinct regulatory mechanisms.