Combination of untargeted and targeted proteomics for secretome analysis of L-WRN cells.

Organoid culture is a promising biomedical technology that requires specialized growth factors. Recently, a recombinant L-WRN cell line has been extensively used to generate conditioned medium (L-CM) for organoid culture. Nevertheless, methods for evaluating the stability of the L-WRN cells have been limited. In this study, a novel proteomics-based approach was developed to analyze the secretome of the cells. Serum-free L-CM was lyophilized, precipitated by trichloroacetic acid, and desalted prior to analysis by liquid chromatography-tandem mass spectrometry. Data-dependent acquisition (DDA) was conducted for the untargeted secretome profiling of the cells, and parallel reaction monitoring (PRM) was applied for the targeted quantification of the Wnt3A, R-spondin3, and noggin proteins (WRNs). This study also compared the performance of two types of PRM methods, namely MS1-independent PRM and MS1-dependent PRM, that can be executed on an Orbitrap instrument. The results showed that the growth of mouse intestinal organoids was closely related to the use of L-CM. The composition of L-CM could be markedly affected by the medium collection scheme. A total of 1725, 2302, and 2681 proteins were identified from the L-CM collected on day 5, day 9, and day 13, respectively. The MS1-independent PRM outperformed the MS1-dependent PRM and effectively quantified the WRNs with high repeatability and specificity. In conclusion, by integrating untargeted and targeted proteomics, this study develops a mass spectrometry-based method for the secretome analysis and quality control of the L-WRN cells. The methodology and findings of the present work will benefit future studies on organoids and secretomes.

Immortalization and Characterization of Rat Lingual Keratinocytes in a High-Calcium and Feeder-Free Culture System Using ROCK Inhibitor Y-27632.

Cultured keratinocytes are desirable models for biological and medical studies. However, primary keratinocytes are difficult to maintain, and there has been little research on lingual keratinocyte culture. Here, we investigated the effect of Y-27632, a Rho kinase (ROCK) inhibitor, on the immortalization and characterization of cultured rat lingual keratinocyte (RLKs). Three Y-27632-supplemented media were screened for the cultivation of RLKs isolated from Sprague-Dawley rats. Phalloidin staining and TUNEL assay were applied to visualize cytoskeleton dynamics and cell apoptosis following Y-27632 removal. Label-free proteomics, RT-PCR, calcium imaging, and cytogenetic studies were conducted to characterize the cultured cells. Results showed that RLKs could be conditionally immortalized in a high-calcium medium in the absence of feeder cells, although they did not exhibit normal karyotypes. The removal of Y-27632 from the culture medium led to reversible cytoskeletal reorganization and nuclear enlargement without triggering apoptosis, and a total of 239 differentially expressed proteins were identified by proteomic analysis. Notably, RLKs derived from the non-taste epithelium expressed some molecular markers characteristic of taste bud cells, yet calcium imaging revealed that they rarely responded to tastants. Collectively, we established a high-calcium and feeder-free culture method for the long-term maintenance of RLKs. Our results shed some new light on the immortalization and differentiation of lingual keratinocytes.

SOX9-COL9A3-dependent regulation of choroid plexus epithelial polarity governs blood-cerebrospinal fluid barrier integrity.

The choroid plexus (CP) is an extensively vascularized neuroepithelial tissue that projects into the brain ventricles. The restriction of transepithelial transport across the CP establishes the blood-cerebrospinal fluid (CSF) barrier that is fundamental to the homeostatic regulation of the central nervous system microenvironment. However, the molecular mechanisms that control this process remain elusive. Here we show that the genetic ablation of Sox9 in the hindbrain CP results in a hyperpermeable blood-CSF barrier that ultimately upsets the CSF electrolyte balance and alters CSF protein composition. Mechanistically, SOX9 is required for the transcriptional up-regulation of Col9a3 in the CP epithelium. The reduction of Col9a3 expression dramatically recapitulates the blood-CSF barrier defects of Sox9 mutants. Loss of collagen IX severely disrupts the structural integrity of the epithelial basement membrane in the CP, leading to progressive loss of extracellular matrix components. Consequently, this perturbs the polarized microtubule dynamics required for correct orientation of apicobasal polarity and thereby impedes tight junction assembly in the CP epithelium. Our findings reveal a pivotal cascade of SOX9-dependent molecular events that is critical for construction of the blood-CSF barrier.